Team:Hamburg/Notebook

Resuspension of required plasmids

Plate	Well	Insert
2017 Kit Plate 3	3F	pSB1C3/BBa_B0015
2017 Kit Plate 4	13L	pSB1A2/BBa_E0040
2017 Kit Plate 2	24D	pSB1C3/BBa_E0840
2017 Kit Plate 3	17C	pSB1C3/BBa_I13504
2017 Kit Plate 3	8P	pSB1C3/BBa_I20270
2017 Kit Plate 3	17L	pSB1C3/BBa_I11032

We pierced through foil with a sterile pipette tip and added 10 µL sterile H₂O. Then we incubated for 10 min at room temperature and transferred 10 µL of the samples into microtubes. Afterward the samples stored at -20°C.

Lotnumber	Part
180202NK01	pSB1C3/BBa_B0015
180202NK02	pSB1A2/BBa_E0040
180202NK03	pSB1C3/BBa_E0840
180202NK04	pSB1C3/BBa_I13504
180202NK05	pSB1C3/BBa_I20270
180202NK06	pSB1C3/BBa_I11032

PCR for amplification of parts by E.coli

Mastermix for 30 samples:

300 µL 5 x Phusion HF Buffer
30 µL dNTPs
30 µL Template DNA (E. coli cells)
15 µL Phusion DNA polymerase
1065 µL H₂O_dest

The new primers were dissolved in sterile H₂O (100 µM). The primer prepared 1:10 dilution (10 µM) and added 1 µL primer to each 48 µL mastermix.

Tube	Part	Fw Primer	Rv Primer
1	MlcRE	Pre-MlcRE-Fw	MlcRE-Suf-Rv
1	MlcRE	MlcRE-Fw	MlcRE-Rv
3	IdhA	Pre-IdhA-Fw	IdhA-Suf
4	IdhA	IdhA-Fw	IdhA-Rv
5	sulA	Pre-sulA-Fw	sulA-Suf-Rv
6	sulA	sulA-Fw	sulA-Rv
7	BBa_B0031_IdhA	31-IdhA-Fw	IdhA-Suf-Rv
8	BBa_B0032_IdhA	32-IdhA-Fw	IdhA-Suf-Rv
9	BBa_B0034_IdhA	34-IdhA-Fw	IdhA-Suf-Rv
10	BBa_B0031_IdhA	31-sulA-Fw	sulA-Suf-Rv
11	BBa_B0032_IdhA	32-sulA-Fw	sulA-Suf-Rv
12	BBa_B0034_IdhA	31-sulA-Fw	sulA-Suf-Rv
13	oHypB Promotor	Pre-HypB-Fw	HypB-Suf-Rv
14	oHypB Promotor	HypB-Fw	HypB-Rv

Annealing:

Tube	Part	Fw Primer	Rv Primer
15	BBa_1718018	dapA-Fw	dapA-Rv

PCR program:

98 °C	3 min
98 °C	10 s
60 °C	30 s
72 °C	40 s
Go to step 2 30x
72 °C	5 min

Annealing program, afterwards chilled on ice:

98 °C	30 s
50 °C	30 s
72 °C	5 min

Restriction

Restriction of pSB1C3_BBa_K20600, pSB1C3_BBa_E0840 and pSB1C3.

	psB1C3_BBa_K206 00	pSB1C3_BBa_E08 40	pSB1C3	pSB1C3
DNA concentration	46.99 ng/µL	57 ng/µL	141 ng/µL	149 ng/µL
DNA	2.13 µL	17.54 µL	21.28 µL	20.13 µL
NE Buffer 2.1	2 µL	5 µL	5 µL	5 µL
Fast AP	-	0.5 µL	0.5 µL	0.5 µL
EcoRI-HF	0.5 µL	0.5 µL	-	1 µL
XbaI	-	0.5 µL	1 µL	-
SpeI	0.5 µL	-	-	-
PstI	-	-	1 µL	1 µL
H 2 O	14.87 µL	25.96 µL	21.22 µL	22.37 µL

The restriction incubated for 80 min at 38°C. After that the restricted plasmids, PCR products and annealing product were stored at -20°C.

PCR for amplification of parts by E.coli

Master mix for 30 preparations:

300 µL 5 x Phusion HF Buffer
30 µL dNTPs
30 µL Template DNA (E. coli cells)
15 µL Phusion DNA polymerase
1065 µL H₂O_dest

Added 1 µL primer to each 48 µL mastermix.

No.	Amplified Part	Fw Primer	Rv Primer
1	MlcRE	Pre- MlcRE-Fw	MlcRE-Suf-Rv
1	MlcRE	MlcRE-Fw	MlcRE-Rv
3	IdhA	Pre-IdhA-Fw	IdhA-Suf
4	IdhA	IdhA-Fw	IdhA-Rv
5	sulA	Pre-sulA-Fw	sulA-Suf-Rv
6	sulA	sulA-Fw	sulA-Rv
7	BBa_B0031_IdhA	31-IdhA-Fw	IdhA-Suf-Rv
8	BBa_B0032_IdhA	32-IdhA-Fw	IdhA-Suf-Rv
9	BBa_B0034_IdhA	34-IdhA-Fw	IdhA-Suf-Rv
10	BBa_B0031_IdhA	31-sulA-Fw	sulA-Suf-Rv
11	BBa_B0032_IdhA	32-sulA-Fw	sulA-Suf-Rv
12	BBa_B0034_IdhA	31-sulA-Fw	sulA-Suf-Rv
13	oHypB Promotor	Pre-HypB-Fw	HypB-Suf-Rv
14	oHypB Promotor	HypB-Fw	HypB-Rv

Annealing:

No.	Amplified Part	Fw Primer	Rv Primer
15	BBa_1718018	dapA-Fw	dapA-Rv

PCR program:

98°C	3 min
98°C	10 s
60°C	30 s
72°C	40 s
Go to step 2 30x
72°C	5 min

Annealing program, afterwards chilled on ice:

98°C	30 s
50°C	30 s
72°C	5 min

Restriction

Restriction of pSB1C3_BBa_K20600, pSB1C3_BBa_E0840 and pSB1C3.

	psB1C3_BBa_K20600	pSB1C3_BBa_E0840	pSB1C3	pSB1C3
DNA concentration	46.99 ng/µL	57 ng/µL	141 ng/µL	149 ng/µL
DNA	2.13 µL	17.54 µL	21.28 µL	20.13 µL
NE Buffer 2.1	2 µL	5 µL	5 µL	5 µL
Fast AP	-	0.5 µL	0.5 µL	0.5 µL
EcoRI-HF	0.5 µL	0.5 µL	-	1 µL
XbaI	-	0.5 µL	1 µL	-
SpeI	0.5 µL	-	-	-
PstI	-	-	1 µL	1 µL
H₂O_dest	14.87 µL	25.96 µL	21.22 µL	22.37 µL

After the restriction the products incubated for 60 min (37°C) and afterwards stored on ice by 4°C.

Restriction

Restriction of pSB1C3 (2x) and pSB1C3_BBa_E0840.

	pSB1C3	pSB1C3	pSB1C3_BBa_E0840
DNA concentration	176.00 ng/µL	155.29 ng/µL	128.00 ng/µL
DNA	17.07 µL	19.32 µL	7.80 µL
NE Buffer 2.1	5.00 µL	5.00 µL	2.00 µL
EcoRI-HF	1.00 µL	-	0.50 µL
XbaI	-	1.00 µL	0.50 µL
SpeI	-	-	-
PstI	1.00 µL	1.00 µL	-
Fast AP	0.50 µL	0.50 µL	0.50 µL
H₂O_dest	25.42 µL	23.18 µL	8.70 µL
ad	49.99 µL	50.00 µL	20.00 µL

Incubated for 60 min by 37°C and afterwards stored on ice by 4°C.

Restriction and ligation

Restriction of vector-parts

Part	pSB1C3	pSB1C3	pSB1C3-BBa_E0840	pSB1C3-K206000
DNA	17.1 µl	17.1 µl	7.8 µl	18 µl
NEBBuffer 2.1	5 µl	5 µl	2 µl	5 µl
EcoRI-HF	1 µl	-	0.5 µl	-
XbaI	-	1 µl	0.5 µl	-
SpeI	-	-	-	0.5 µl
PstI	1 µl	1 µl	-	0.5 µl
FastAP	0.5 µl	0.5 µl	0.5 µl	0.5 µl
ddH₂O	25.4 µl	25.4 µl	8.7 µl	25.5 µl
ad	50 µl	50 µl	20 µl	50 µl

The samples were incubated at 37°C for 1 h and following 20 min at 80°C. Samples were applied on a 1.5% agarose gel and extracted through the “GeneJet Gel Extraction Kit” of Thermo Fisher.

Restiction of the PCR products

Part	MlcRE (1)	MlcRE (2)	sulA	BBa_B0031-sulA	BBa_B0032-sulA	BBa_B0034-sulA	oHybB (1)	oHybB (2)
DNA	10 µl	10 µl	20 µl	20 µl	20 µl	20 µl	20 µl	20 µl
NEBBuffer 2.1	2 µl	2 µl	5 µl	5 µl	5 µl	5 µl	5 µl	5 µl
EcoRI-HF	0.5 µl	0.5 µl	0.5 µl	-	-	-	0.5 µl	0.5 µl
XbaI	-	-	-	0.5 µl	0.5 µl	0.5 µl	-	-
SpeI	-	0.5 µl	-	-	-	-	-	0.5 µl
PstI	0.5 µl	-	0.5 µl	0.5 µl	0.5 µl	0.5 µl	0.5 µl	-
FastAP	-	-	-	-	-	-	-	-
ddH₂O	7 µl	7 µl	24 µl	24 µl	24 µl	24 µl	24 µl	24 µl
ad	20 µl	20 µl	50 µl	50 µl	50 µl	50 µl	50 µl	50 µl

The samples were incubated at 37°C for 1 h and following 20 min at 80°C.

Ligation

Product	pSB1C3-MlcRE(1)	pSB1C3-MlcRE(2)-BBa_E0840	pSB1C3-sulA	pSB1C3-BBa_B0031-sulA	pSB1C3-BBa_B0032-sulA
Vector	3.01 µl	7.7 µl	1.86 µl	1.97 µl	1.97 µl
Part	8.25 µl	7.09 µl	15.18 µl	9.48 µl	13.07 µl
T4 Buffer	2 µl	2 µl	2 µl	2 µl	2 µl
T4 Ligase	0.5 µl	0.5 µl	0.5 µl	0.5 µl	0.5 µl
ddH₂O	6.24 µl	2.71 µl	0.46 µl	6.06 µl	2.46 µl
ad	20 µl	20 µl	20 µl	20 µl	20 µl

Product	pSB1C3-BBa_B0034-sulA	pSB1C3-oHybB(1)	pSB1C3-oHybB(2)-E0840	pSB1C3-oHybB(2)
Vector	1.97 µl	3.29 µl	8.52 µl	3.29 µl
Part	22.56 µl	9.21 µl	8.26 µl	9.94 µl
T4 Buffer	5 µl	2 µl	2 µl	2 µl
T4 Ligase	0.5 µl	0.5 µl	0.5 µl	0.5 µl
ddH₂O	19.97 µl	4.99 µl	0.72 µl	4.27 µl
ad	50 µl	20 µl	20 µl	20 µl

The samples were incubated at 16°C overnight.

Colony PCR of the transformation performed on the 22^nd of March

Mastermix

Substance	1x (volume in µl)	60x (volume in µl)
10x DreamTaq Buffer	2	120
dNTPs 10mM	2	120
MgCl₂ 50mM	1.2	72
Fw-Primer VF2	0.6	36
Rv-Primer VR	0.6	36
Taq-Polymerase	0.1	6
ddH₂O ad 20µl	13.5	810

Colonies were picked with pipette-tips and transferred into 1 ml LB-Medium tubes after they were put in 20 µl aliquots of the mastermix of the following PCR reaction

PCR-Programm (30 cycles)

Temperature (°C)	Time (s)
95	180
94	30
55	30
72	90
72	600

PCR products were stored at 4°C over the weekend, no agarose gel was performed.

Repetition of the colony PCR performed on the 29^th of March

The colony PCR perfomed on the 29^th of March had been applied on an 1% agarose gel but since the DNA-ladder had been forgotten the colony PCR was repeated.

Mastermix

Substance	1x (volume in µl)	60x (volume in µl)
10x DreamTaq Buffer	2	120
dNTPs 10mM	2	120
MgCl₂ 50mM	1.2	72
Fw-Primer VF2	0.6	36
Rv-Primer VR	0.6	36
Taq-Polymerase	0.1	6
ddH₂O ad 20µl	13.5	810

Colonies were picked with pipette-tips and transferred into 1 ml LB-Medium tubes after they were put in 20 µl aliquots of the mastermix of the following PCR reaction

PCR-Programm (30 cycles)

Temperature (°C)	Time (s)
95	180
94	30
55	30
72	90
72	600

The bands were accidentally cut out and purified by using the “GeneJet Gel Extraction Kit” from Thermo Fisher.

PCR of multiple parts.

PCR were prepapared as followed:

Tube	Part	Primer-fw	Primer-rv
1	IdhA	Pre_IdhA-Fw	IdhA-Suf-Rev
2	IdhA	IdhA-Fw	IdhA-Rev
3	BBa_B0031_IdhA	31-Idha-Fw	IdhA-Suf-Rev
4	BBa_B0032_IdhA	32-Idha-Fw	IdhA-Suf-Rev
5	BBa_B0034_IdhA	34-Idha-Fw	IdhA-Suf-Rev
6	BBa_B0032_sulA	32-sulA-Fw	SulA-Suf-Rev
7	oHybB	Pre-HybB-Fw	HybB-Suf-Rev
8	oHybB	HybB-Fw	HybB-Rev
9 (Annealing)	BBa_I718018	dapAp-Fw	dapAp-Rev

Mastermix for 15 approaches were prepared as shown:

150 µL	5x Phusion HF Buffer
15 µL	dNTPs
15 µL	Template DNA (DH5α)
7.5 µL	Phusion DNA Polymerase
532.5 µL	H₂O

This was aliquoted into 0.5 mL reaction tube to total volume of 48 µL. To each aliquot were added the specific primer pair, 1 µL forward and 1 µL reverse primer.

The PCR program was done as shown in the next table.

1	98 °C	3 min
2	98 °C	10 s
3	60 °C	30 s
4	72 °C	60 s
5	go to step 2 30 times
6	72 °C	5 min

For the Annealing we used the following program.

1	98 °C	30 s
2	50 °C	30 s
3	60 °C	5 min

Restriction of pSB1C3 and BBa_K206000

For the digestion of pSB1C3 we used two approaches and for BBa_K206000 one approach as shown in the following table.

	pSB1C3 (R1)	pSB1C3 (R2)	BBa_K206000 (R3)
DNA content	155.29 ng/µL	155.29 ng/µL	140.31 ng/µL
DNA used	19.32 µL	19.32 µL	19.00 µL
NE-Buffer 2.1	5.00 µL	5.00 µL	5.00 µL
FAST-Ap	0.50 µL	0.50 µL	0.50 µL
EcoRI-HF	1.00 µL	0.50 µL	-
XbaI	-	1.00 µL	-
PstI	1.00 µL	1.00 µL	0.50 µL
SpeI	-	-	0.50 µL
H₂O	23.18 µL	23.18 µL	25.5 µL

The restriction digests were incubated for 1 hour at 37 °C.

With the gel electroporation the correctness for the PCR and restriction can be shown. In this approach the PCR of Tube 6, 7 and 8 were correct and the restriction digest of all three constructs worked. For this 6 constructs a gel extraction with the Thermo Scientific Gel extraction Kit was done and the concentration was measured with a nanodrop.

Probe	Concentration
Tube 6	17.863 ng/µL
Tube 7	29.722 ng/µL
Tube 8	30.555 ng/µL
R1	24.91 ng/µL
R2	6.813 ng/µL
R3	30.245 ng/µL

Miniprep of a colony PCR

For the miniprep of the colony PCR products these listed parts were used. The miniprep was done with the Kit from Thermo Scientific protocol A. The last centrifugation step was done by 8600 rpm for 2 min. The concentration was measured with a nanodrop.

Lot number	Concentration
180329DW02	72.183 ng/µL
180329DW03	101.987 ng/µL
180329DW05	9.721 ng/µL
180329DW06	9.051 ng/µL
180329DW09	131.545 ng/µL
180329DW10	98.964 ng/µL
180329DW14	25.731 ng/µL
180329DW15	26.956 ng/µL
180329DW21	75.207 ng/µL
180329DW23	76.729 ng/µL
180329DW26	18.672 ng/µL
180329DW27	31.688 ng/µL
180329DW28	139.269 ng/µL
180329DW30	9.514 ng/µL
180329DW39	93.788 ng/µL
180329DW41	101.325 ng/µL
180329DW44	101.745 ng/µL
180329DW45	17.186 ng/µL

The Parts were stored at -20 °C

Repetition PCR ldHA/Annealing

Tube	Part	Primer
1	ldhA	Pre-ldhA-Fw + ldhA-suf-Rv
2	ldhA	ldhA-Fw + ldhA-Rv
3	BBa_B0031_ldhA	31-LdhA-Fw + ldhA-suf-Rv
4	BBa_B0032_ldhA	32-ldhA-Fw + ldhA-suf-Rv
5	BBa_B0034_ldhA	34-ldhA-Fw + ldhA-suf-Rv
6	BBa_I718018	dapAp-Fw + dapAp-Rv

Mastermix for 15 uses:

150 µL 5x Phusion HF Buffer
15 µL dNTPs
15 µL Template DNA (E.coli cells DH5a)
5 µL Phusion DNA Polymerase
5 µL H₂O_dest

Mastermix 48 µL + each 1 µL Primer

PCR-Programm:

98°C	3 min
98°C	10 s
60°C	30 s
72°C	90 s
GOTO Step 2 30x
72°C	5 min

Ligation-Programm

98°C	3 min
98°C	30 s
50°C	30 s
72°C	5 min
GOTO Step 2 30x
72°C	5 min

Restriction of BBa_B0032_sulA and oHybB (Pre + Suffix)

	BBa_B0032_sulA	oHybB	oHybB
DNA	5.6 µL	3.36 µL	3.33 µL
NE 2.1	2 µL	2 µL	2 µL
EcoR1	-	0.5 µL	0.5 µL
Xba1	0.5 µL	-	-
Spe1	-	-	0.5 µL
Pst1	0.5 µL	0.5 µL	-
FastAp	-	-	-
H₂O	11.4 µL	13.64 µL	13.67 µL
Total	20 µL	20 µL	20 µL

Incubated for 1 h by 37°C and stored at -20°C.

180406DK01 = BBa_B0032_sulA
180406DK02 = oHybB cut with EcoR1 and Pst1
180406DK03 = OHybB cut with EcoR1 and Spe1

PCR of BBa_B0031SulA, BBa_1718018 (Annealing)

After the scheme on page 18 a new Mastermix was pipetted together

Tube	Part	Primer
1	BBa_B0031SulA	31-SulA-FW + SulA-Suf-RV
2	BBa_1718018	DapAP-FW + DapAP-RV

48 µL Mastermix per 1 µL Primer was used.

PCR-Program

98 °C	3 min
98 °C	10 s
60 °C	30 s
72 °C	90 s
GOTO Step 2 30 x
72 °C	5 min

Annealing-Program

98 °C	30 s
50 °C	40 s
72°C	5 min

Restriction of LdhA

Name	LdhA	BBa_B0031_LdhA	BBa_0032_LdhA	BBa_0034_LdhA
NE 2.1 buffer	40 µL	40 µL	40 µL	40 µL
EcoRI	0.5 µL	-	-	-
XbaI	-	0.5 µL	0.5 µL	0.5 µL
SpeI	-	-	-	-
PstI	0.5 µL	0.5 µL	0.5 µL	0.5 µL
Fast AP	-	-	-	-
H₂O dest.	4 µL	4 µL	4 µL	4 µL
ges.	50 µL	50 µL	50 µL	50 µL

Incubatated at 27 °C for 1h

LdhA	180409NK01
31LdhA	180409NK02
32LdhA	180409NK03
34LdhA	180409NK04

Ligation of pSB1C3-IdhA, pSB1C3-BBa_0031-IdhA, pSB1C3-BBa_B0032-IdhA and pSB1C3-BBa_B0034-IdhA

pSB1C3(1) was cut with EcoR1-Hf and PstI; pSB1C3(2) was cut with XbaI and PstI

Construct: pSB1C3-IdhA

Substance	Volume (µL)
Vector pSB1C3 (1)	0.67
Part IdhA	3.35
T4 Buffer	2.00
T4 Ligase	0.50
H₂O	13.45

Total: 20 µL Ligated part name: 180412NK01

Construct: pSB1C3-BBa_B0031-IdhA

Substance	Volume (µL)
Vector pSB1C3 (2)	2.49
Part BBa_B0031-IdhA	5.72
T4 Buffer	2.00
T4 Ligase	0.50
H₂O	9.35

Total: 20 µL Ligated part name: 180412NK02

Construct: pSB1C3-BBa_B0032-IdhA

Substance	Volume (µL)
Vector pSB1C3 (2)	2.49
Part BBa_B0032-IdhA	5.72
T4 Buffer	2.00
T4 Ligase	0.50
H₂O	9.36

Total: 20 µL Ligated part name: 180412NK03

Construct: pSB1C3-BBa_B0034-IdhA

Substance	Volume (µL)
Vector pSB1C3 (2)	2.43
Part BBa_B0034-IdhA	4.77
T4 Buffer	2.00
T4 Ligase	0.50
H₂O	10.30

Total: 20 µL Ligated part name: 180412NK04

Each ligated part was incubated at 37°C for 1h and was stored at -20°C.

Repetition of the PCR of MlcRE with Pre and Suffix

Mastermix for (30 cycles) was prepared:

Substance	Volume (µL)
5x Phusion HF Buffer	300
dNTPs	30
Template DNA (E. coli cells)	30
Phusion DNA Polymerase	15
H₂O	1065

Primers:

Substance	Volume (µL)
dapAP-Fw	1
dapAP-Rev	1

For each PCR 48µL of the Mastermix and 1µL of the pair of primers was used.

PCR-Programm (30 cycles)

Temperature (C°)	Time (s)
98	180
98	10
60	30
72	90
72	300

Ligation of pSB1C3-MIcRE-BBa_B0031-IdhA, pSB1C3-MlcRE-BBa_B0032-idhA, pSB1C3-MlcRE-BBa-B0034-IdhA

Each Ligation had a total Volume of 20 µL and was run at 16 °C overnight.

L3:

Construct: pSB1C3-MlcRE-BBa_B0031-IdhA (180417AK01)

Substance	Volume (µL)
Vector pSB1C3 (1)	0.55
Part 1: MlcRE	2.07
Part 2: 31 IdhA	4.76
T4 Buffer	2.00
T4 Ligase	0.50
H₂O_dest	10.12

L4:

Construct: pSB1C3-MlcRE-BBa_B0032-IdhA (180417AK02)

Substance	Volume (µL)
Vector pSB1C3 (1)	0.55
Part 1: MlcRE	2.07
Part 2: 32 IdhA	4.76
T4 Buffer	2.00
T4 Ligase	0.50
H₂O_dest	10.12

L5:

Construct: pSB1C3-MlcRE-BBa_B0031-IdhA (180417AK03)

Substance	Volume (µL)
Vector pSB1C3 (1)	0.55
Part 1: MlcRE	2.07
Part 2: 34 IdhA	3.97
T4 Buffer	2.00
T4 Ligase	0.50
H₂O_dest	10.90

Repetition of Ligations L12, L16 and L5

Each Ligation had a total Volume of 20 µL and was run for 1h, 400 rpm at 37 °C.

L5:

Construct: pSB1C3-MlcRE-BBa_B0034-IdhA (180425LD01)

Substance	Volume (µL)
Vector	0.55
Part 1	2.07
Part 2	3.97
T4 Buffer	2.00
T4 Ligase	0.50
H₂O_dest	10.09

L12:

Construct: pSB1C3-BBa_B0032-SulA (180425LD02)

Substance	Volume (µL)
Vector	3.94
Part 1	6.00
Part 2	-
T4 Buffer	2.00
T4 Ligase	0.50
H₂O_dest	7.50

L16:

Construct: pSB1C3-oHybB (180425LD03)

Substance	Volume (µL)
Vector	1.93
Part 1	1.14
Part 2	-
T4 Buffer	2.00
T4 Ligase	0.50
H₂O_dest	14.43

Transformation of the ready parts for the cryostocks (characterizations)

Protocol was by Zhang Ghong.

Lot number plasmid	Part	Lot number culture
180329DW03	pSB1C3-MlcRE	180517LD01
180329DW05	pSB1C3-MlcRE-BBa_E0840	180517LD02
180329DW09	pSB1C3-sulA	180517LD03
180329DW14	pSB1C3-BBa_B0031_sulA	180517LD04
180329DW21	pSB1C3-BBa_B0034_suA	180517LD05
180329DW27	pSB1C3-oHybB	180517LD06
180329DW28	pSB1C3-oHybB_BBa_E0840	180517LD07
180329DW41	pSB1C3-BBa_K20600_BBa_B0032_sulA	180517LD08
180329DW45	pSB1C3- BBa_K20600_BBa_B0034_sulA	180517LD09
180426NK20	pSB1C3-BBa_B0032-LdhA	180517LD10
180426NK28	pSB1C3- BBa_B0034-LdhA	180517LD11
180426NK30	pSB1C3-BBa_K20600_BBa_B0031_sulA	180517LD12
180509NF01	pSB1C3-BBa_I20270	180517LD13
180508OM01	pSB1C3-BBa_B0030	180517LD14

Setting up cryotocks

Lot number culture	Part	Lot number cryostock
180517LD01	pSB1C3-MlcRE	180518BK01
180517LD02	pSB1C3-MlcRE-BBa_E840	180518BK02
180517LD03	pSB1C3-sulA	180518BK03
180517LD04	pSB1C3-BBa_B0031_sulA	180518BK04
180517LD05	pSB1C3- BBa_B0034_sulA	180518BK05
180517LD06	pSB1C3-oHybB	180518BK06
180517LD07	pSB1C3-oHybB-BBa_E0840	180518BK07
180517LD08	pSB1C3-BBa_K206000-BBa_B0032-sulA	180518BK08
180517LD09	pSB1C3-BBa_K206000-BBa_B0034-sulA	180518BK09
180517LD10	pSB1C3-BBa_B0032-sulA	180518BK10
180517LD11	pSB1C3-BBa_B0034-sulA	180518BK11
180517LD12	pSB1C3-BBa_K20600-BBa_B0031-sulA	180518BK12
180517LD13	pSB1C3-BBa_I20270	180518BK13
180508OM06	pSB1C3-BBa_B0031	180518BK14
180508OM09	pSB1C3-BBa_B0034	180518BK15
180508OM08	pSB1C3-BBa_B0033	180518BK16
180508OM10	pSB1C3-BBa_K554008	180518BK17
180508OM12	pSB1C3-BBa_K554002	180518BK18
180508OM11	pSB1C3-BBa_K554009	180518BK19
180508OM07	pSB1C3-BBa_B0032	180518BK20

600 µL of the culture medium were mixed with 200 µL 50% Glycerol and put them into tubes. Frozen the tubes in liquid nitrogen and stored at -80°C.

PCR of the new Parts

Mastermix for 20 samples:

200 µL 5x HF Buffer
20 µL dNTPs
10 µL Phusion DNA-Polymerase
710 µL H₂O_dest
20 µL Template DNA (DH5α)

PCR program:

98°C	3 min
98°C	10 s
60°C	30 s
72°C	90 s
Go to step 2 30x
72°C	5 min

PCR products:

Tube	Part	Length
1	ldhA	1036
2	BBa_B0034-ldhA	1054
3	BBa_B0031-ldhA	1056
4	BBa_B0032-ldhA	1055
5	BBa_B0032-sulA	575
6	BBa_J45503	446
7	cspD	273
8	accA	1011
9	accB	522
10	tesA	678
11	cbtA	426
12	mraz	507
13	leuABCD	4748
14	leuABCD6H	4766
15	OmpT	1002
16	MlcRE	252

180522- 2

Gelelectrophoresis

We made a 1% agarose gel.

Liquid cultures for characterizations

10 mL Lb-Cmp liquid cultures were inoculated with colonies from 108522 plates:

Plate	Part
180522NF01	pSB1C3-MlcRE-BBa_E0840
180522NF02	pSB1C3-oHybB-BBa_E0840
180522NF03	pSB1C3-BBa_K206000-BBa_B0032-sulA
180522NF04	pSB1C3-BBa_K206000-BBa_B0034-sulA
180522NF05	pSB1C3-BBa_K206000-BBa_B0031-sulA

Additionally, cryostock of pSB1C3-BBa_I20270 was used for an additional liquid culture.

180523 – 1

Restriction and ligation of multiple parts

Restriction:

Tube

1

2

3

4

5

6

7

8

9

10

11

Part

pSB1C3-BBa_B0031

pSB1C3-BBa_B0032

pSB1C3-BBa_B0033

pSB133-BBa_E0840

Bba_B0032_sulA

Bba_J45503

cspD

accB

cbtA

mraZ

pSB1C3-I20270

Lot#

180514NB01

180514NB07

180514NB03

180315NB01

aus PCR

aus PCr

aus PCR

180509NF01

ρ

153 ng/µL

81 ng/µL

128 ng/µL

31 ng/µL

24 ng/µL

26 ng/µL

37 ng/µL

33 ng/µL

166 ng/µL

Use

target m

150 ng

100 ng

150 ng

250 ng

100 ng

300 ng

2000 ng

Volumes

DNA

0.98 µL

1.86 µL

1.85 µL

0.78 µL

3.22 µL

6.28 µL

9.79 µL

2.71 µL

8.06 µL

9.10 µL

12.05 µL

NEBuffer 2.1

1.00 µL

2.00 µL

1.00 µL

2.00 µL

EcoRI-HF

0.50 µL

XbaI

0.50 µL

SpeI

0.50 µL

PstI

0.50 µL

FastAP

0.50 µL

H₂O

6.52 µL

5.64 µL

5.65 µL

6.72 µL

4.78 µL

1.72 µL

7.21 µL

5.29 µL

8.94 µL

7.90 µL

4.45 µL

ad

10.00 µL

20.00 µL

10.00 µL

20.00 µL

Länge Produkt

ρ final

15 ng/µL

10 ng/µL

15 ng/µL

13 ng/µL

10 ng/µL

15 ng/µL

100 ng/µL

Ligation:

Tube

1

2

3

4

5

6

7

8

9

10

11

Product

pSB1C3-Bba_B0032-sulA

pSB1C3-Bba_J45503-Bba_E0840

pSB1C3-BBa_B0031-cspD

pSB1C3-BBa_B0032-cspD

pSB1C3-Bba_B0033-cspD

pSB1C3-cspD

pSB1C3-Bba_B0032-cbtA

pSB1C3-Bba_B0033-cbtA

pSB1C3-cbtA

pSB1C3-Bba_B0033-mraZ

pSB1C3-mraZ

Vector

Vector

pSB1C3-I20270

pSB1C3-BBa_E0840

pSB1C3-BBa_B0031

pSB1C3-BBa_B0032

pSB1C3-BBa_B0033

pSB1C3-I20270

pSB1C3-BBa_B0032

pSB1C3-BBa_B0033

pSB1C3-I20270

pSB1C3-BBa_B0033

pSB1C3-I20270

Tube

11

4

1

2

3

11

2

3

11

3

11

ρ

24 ng/µL

5 ng/µL

4 ng/µL

36 ng/µL

24 ng/µL

4 ng/µL

36 ng/µL

24 ng/µL

36 ng/µL

24 ng/µL

Länge

2989 bp

2948 bp

2084 bp

2083 bp

2081 bp

2989 bp

2038 bp

2081 bp

2989 bp

2081 bp

2989 bp

V

1.43 µL

8.65 µL

10.69 µL

9.84 µL

1.21 µL

2.19 µL

7.46 µL

0.92 µL

1.73 µL

0.81 µL

1.55 µL

Part 1

Part 1

Bba_B0032_sulA

Bba_J45503

cspD

cbtA

mraZ

Tube

5

6

7

9

10

ρ

10 ng/µL

15 ng/µL

13 ng/µL

15 ng/µL

Length

575 bp

446 bp

273 bp

426 bp

507 bp

V

6.59 µL

4.01 µL

4.36 µL

3.67 µL

4.48 µL

3.92 µL

4.73 µL

4.19 µL

Part 2

Part 2

Tube

ρ

Length

V

T4 Buffer

1.00 µL

2.00 µL

1.00 µL

2.00 µL

1.00 µL

T4 Ligase

0.50 µL

H2O

0.48 µL

4.84 µL

2.45 µL

3.30 µL

2.93 µL

2.64 µL

5.56 µL

3.11 µL

2.85 µL

2.96 µL

2.76 µL

ad

10.00 µL

20.00 µL

10.00 µL

20.00 µL

10.00 µL

Transformation

Competent DH5a cells were thawed at 4°C on ice for 5 min. Cells were inoculated with Ligations from 180523 and incubated for 1 h at 4°C on ice. Heat shock was performed at 42°C for 60 s. Cells were chilled at 4°C on ice for 5 min. 850 µL LB medium was added, and cells were incubated at 37°C, 900 RPM on the thermomix for 1h. Cells were brought onto LB-agar-Cmp plates and incubated at 37°C overnight as:

Part	Plate
pSB1C3-BBa_B0032-sulA	170524NB01
pSB1C3-BBa_J45503-BBa_E0840	170524NB02
pSB1C3-BBa_B0031-cspD	170524NB03
pSB1C3-BBa_B0032-cspD	170524NB04
pSB1C3-BBa_B0033-cspD	170524NB05
pSB1C3-cspD	170524NB06
pSB1C3-BBa_B0032-cbtA	170524NB07
pSB1C3-BBa_B0033-cbtA	170524NB08
pSB1C3-cbtA	170524NB09
pSB1C3-BBa_B0033-mraZ	170524NB10
pSB1C3-mraZ	170524NB11

Characterization of ohybB and MlcRE

Set up precultures and incubated at 37°C and 210 rpm.

OD-measure at 11:30 am.

Part	OD
ohybB (4°C)	0.752
MlcRe (0g/L)	0.307
DH5α (negative control)	0.236
GFP (positive control)	0.385
MlcRE (0.125 g/L)	0.391
MlcRE (0.25 g/L)	0.358
MlcRE (0.5 g/L)	0.382
MlcRE (1 g/L)	0.427
MlcRE (2 g/L)	0.418

Part	Temperature (°C)	OD
ohybB	10	0.727
ohybB	15	0.746
ohybB	20	0.709
ohybB	25	0.729
ohybB	30	0.782
ohybB	37	0.831

The ohybB series were diluted 1:1. All cultures were transferred at 4°C. Of the cultures MlcRE, DH5α and GFP were prepared new cultures. To all cultures were 6.5 mL LB-medium added. After another OD-measurement after 1 h the measure were cancelled. The culture tubes stored at 4°C over night.

Colony PCR of 180524 transformation

Colonies of 180524 transformations were picked and, and colony PCR was set up employing a PCR master mix for 30 PCRs in 20 µL each:

DreamTaq Buffer	8 µL
dNTPs 25 mM each	16 µL
50 mM MgCl₂	24 µL
10 µM VF2	24 µL
10 µM VR	24 µL
DreamTaq	1 µL
H₂O	632 µL
ad	800 µL

20 µL master mix was inoculated with a colony of 180524 transformation plates. PCR was carried out as follows:

1	95°C	3:00
2	94°C	0:30
3	55°C	0:30
4	72°C	1:30
5	GOTO 2, rep 30x
6	72°C	5:00
8	4°C	Hold

PCR of E. coli genomic basic parts

Basic parts were amplified from E. coli genomic DNA. A PCR master mix was set up as following:

5x Phusion HF Buffer	200 µL
dNTPs	20 µL
Phusion DNA Polymerase	10 µL
H₂O	710 µL
DNA Template	20 µL

50 µL PCRs were set up using 48 µL master mix plus 1 µL forward and reverse primers each:

Product	Forward primer	Reverse primer
BBPre-ldhA-BBSuf	Pre_ldhA_f	ldhA_Suf_r
XbaI-BBa_B0034-ldhA-BBSuf	34_ldhA_f	ldhA_Suf_r
XbaI-BBa_B0031-ldhA-BBSuf	31_ldhA_f	ldhA_Suf_r
XbaI-BBa_B0032-ldhA-BBSuf	32_ldhA_f	ldhA_Suf_r
BBPre-BBa_J45503-BBSuf	Pre_hybB_f	hybB_Suf_r
BBPre-cspD-BBSuf	Pre_cspD_f	cspD_Suf_r
BBPre-accA-BBSuf	Pre_accA_f	accA_Suf_r
BBPre-accB-BBSuf	Pre_accB_f	accB_Suf_r
BBPre-tesA-BBSuf	Pre_tesA_f	tesA_Suf_r
BBPre-cbtA-BBSuf	Pre_cbtA_f	cbtA_Suf_r
BBPre-mraZ-BBsuf	Pre_mraZ_f	mraZ_Suf_r
BBPre-LeuABCD-BBSuf	Pre_LeuA_f	LeuD_Suf_r
BBPre-LeuABCD-His-BBSuf	Pre_LeuA_f	LeuD_His_Suf_r
BBPre-OmpT-BBSuf	Pre_OmpT_f	OmpT_Suf_r
BBPre-MlcRE-BBSuf	Pre_MlcRE_f	MlcRE_Suf_r
BBPre-BBa_B0031-ldhA-BBSuf (amplified with Taq)	31_ldhA_f	ldhA_Suf_r

PCR was carried out using following protocol:

1	98°C	3:00
2	98°C	0:10
3	60°C	0:30
4	72°C	2:00
5	GOTO 2, rep 30x
6	72°C	5:00

PCR products were analysed by agarose gel electrophoresis:

Correctly amplified products were purified using GeneJet PCR Purification Kit (ThermoFisher) and stored as:

180604NB01	BBa_J45503
180604NB02	cspD
180604NB03	cbtA
180604NB04	mraZ
180604NB05	MlcRE
180604NB06	BBa_B0031-ldhA

PCR of the 2nd Generation from e. coli

All PCR Reactions were prepared as written below.

19 µL	H₂O
1 µL	DH5α
2.5 µL	Fw-Primer
2.5 µL	Rv-Primer
25 µL	Q5 High-Fidelity 2X Master Mix

Tube	Part	Fw-Primer	Rv-Primer	Predicted length (bp)	Concentration (ng/µL)
1	ldhA	Pre-ldhA-fw	ldhA-Suf-rv	1036	34.465
2	BBa_B0034-ldhA	34-ldhA-fw	ldhA-Suf-rv	1054	43.920
3	BBa_B0031-ldhA	31-ldhA-fw	ldhA-Suf-rv	1056	40.609
4	BBa_B0032-ldhA	32-ldhA-fw	ldhA-Suf-rv	1055	39.545
5	BBa_J45503	Pre-45503-fw	45503-Suf-rv	446	41.660
6	cspD	Pre-cspD-fw	cspD-Suf-rv	273	46.327
7	accA	Pre-accA-fw	accA-Suf-rv	1011	35.789
8	accB	Pre-accB-fw	accB-Suf-rv	522	X
9	tesA	Pre-tesA-fw	tesA-Suf-rv	678	38.747
10	cbtA	Pre-cbtA-fw	cbtA-Suf-rv	426	50.447
11	mraZ	Pre-mraZ-fw	mraZ-Suf-rv	507	X
12	LeuABCD	Pre-LeuA-fw	LeuD-Suf-rv	4748	X
13	LeuABCD6H	Pre-LeuA-fw	LeuD6H-Suf-rv	4766	X
14	OmpT	Pre-OmpT-fw	OmpT-Suf-rv	1002	X
15	MlcRE	Pre-MlcRE-fw	MlcRE-Suf-rv	252	54.880

PCR was performed using following Cycler-Protocol

Temperature (°C)	Time	Time (for LeuABCD)	Repeats
98	2:00	2:00	1
98	0:10	0:10	30
60	0:30	0:30	30
72	1:00	3:00	30
72	2:00	2:00	1
4	∞	∞	1

A gel electrophoresis was performed using a 1%-Agarose-Gel, for LeuABCD a 0.7%-Agarose-Gel was prepared but got destroyed due to high voltage.

PCR Purification was performed using the Thermo Scientific PCR Purification Kit. The PCR Products were stored at 4 °C.

Restriction of PCR Products

The reaction was prepared as seen in the table below, incubated at 37 °C for 60 minutes and heat inactivated at 80 °C for 20 minutes.

Tube

1

2

4

5

6

7

8

9

10

11

12

13

14

15

16

17

18

19

Part

pSB1C3-BBa_B0031

pSB1C3-BBa_B0032

pSB133-BBa_K206000

pSB1C3-I20270

pSB1C3-BBa_E0840

pSB1C3-I20270

BBa_J45503

cspD

accA

accB

cbtA

mraZ

ldhA

BBa_B0031-ldhA

BBa_B0032-ldhA

BBa_B0034-ldhA

MlcRE

Lot#

180514NB01

180514NB07

180321NB01

180509NF01

180315NB01

180509NF01

aus PCR

ρ

153 ng/µL

81 ng/µL

140 ng/µL

166 ng/µL

128 ng/µL

166 ng/µL

42 ng/µL

46 ng/µL

30 ng/µL

36 ng/µL

39 ng/µL

50 ng/µL

34 ng/µL

41 ng/µL

40 ng/µL

44 ng/µL

55 ng/µL

Use

target m

200 ng

500 ng

200 ng

100 ng

200 ng

150 ng

300 ng

250 ng

200 ng

100 ng

Volumes

DNA

1.31 µL

2.48 µL

1.43 µL

6.02 µL

0.60 µL

0.78 µL

0.60 µL

4.80 µL

4.32 µL

3.39 µL

5.59 µL

3.87 µL

5.95 µL

7.30 µL

10.00 µL

2.00 µL

NEBuffer 2.1

1.00 µL

2.00 µL

1.00 µL

2.00 µL

1.00 µL

EcoRI-HF

0.50 µL

XbaI

0.50 µL

SpeI

0.50 µL

PstI

0.50 µL

FastAP

0.50 µL

H2O

6.19 µL

5.02 µL

6.07 µL

10.48 µL

6.90 µL

6.72 µL

6.90 µL

3.20 µL

3.68 µL

4.61 µL

2.41 µL

4.13 µL

2.05 µL

9.70 µL

7.00 µL

6.00 µL

ad

10.00 µL

20.00 µL

10.00 µL

20.00 µL

10.00 µL

Länge Produkt

ρ final

20 ng/µL

50 ng/µL

10 ng/µL

20 ng/µL

10 ng/µL

20 ng/µL

15 ng/µL

30 ng/µL

13 ng/µL

20 ng/µL

22 ng/µL

11 ng/µL

The gel for the vectors melted. Restrictions needed to be repeated.

Repetition of the 180614-Restriction

The reaction was prepared as seen in the table below, incubated at 37 °C for 60 minutes and heat inactivated at 80 °C for 20 minutes.

Tube

1

2

4

5

6

7

8

9

10

11

12

13

14

15

16

17

18

19

Part

pSB1C3-BBa_B0031

pSB1C3-BBa_B0032

pSB133-BBa_K206000

pSB1C3-I20270

pSB1C3-BBa_E0840

pSB1C3-I20270

BBa_J45503

cspD

accA

accB

cbtA

mraZ

ldhA

BBa_B0031-ldhA

BBa_B0032-ldhA

BBa_B0034-ldhA

MlcRE

Lot#

180514NB01

180514NB07

180321NB01

180509NF01

180315NB01

180509NF01

aus PCR

ρ

153 ng/µL

81 ng/µL

140 ng/µL

166 ng/µL

128 ng/µL

166 ng/µL

42 ng/µL

46 ng/µL

30 ng/µL

36 ng/µL

39 ng/µL

50 ng/µL

34 ng/µL

41 ng/µL

40 ng/µL

44 ng/µL

55 ng/µL

Use

target m

200 ng

500 ng

200 ng

100 ng

200 ng

150 ng

300 ng

250 ng

200 ng

100 ng

Volumes

DNA

1.31 µL

2.48 µL

1.43 µL

6.02 µL

0.60 µL

0.78 µL

0.60 µL

4.80 µL

4.32 µL

3.39 µL

5.59 µL

3.87 µL

5.95 µL

7.30 µL

10.00 µL

2.00 µL

NEBuffer 2.1

1.00 µL

2.00 µL

1.00 µL

2.00 µL

1.00 µL

EcoRI-HF

0.50 µL

XbaI

0.50 µL

SpeI

0.50 µL

PstI

0.50 µL

FastAP

0.50 µL

H2O

6.19 µL

5.02 µL

6.07 µL

10.48 µL

6.90 µL

6.72 µL

6.90 µL

3.20 µL

3.68 µL

4.61 µL

2.41 µL

4.13 µL

2.05 µL

9.70 µL

7.00 µL

6.00 µL

ad

10.00 µL

20.00 µL

10.00 µL

20.00 µL

10.00 µL

Länge Produkt

ρ final

20 ng/µL

50 ng/µL

10 ng/µL

20 ng/µL

10 ng/µL

20 ng/µL

15 ng/µL

30 ng/µL

13 ng/µL

20 ng/µL

22 ng/µL

11 ng/µL

24

15

13 ng/µL

10

15

For the Restrictions 1-7 a 1%-Agarose gel was prepared and 4, 5 and 7 were extracted using the Thermo Scientific Gel Extraction Kit.

The concentration of tube 4 was 4.03 ng/µL, the concentration of tube 5 was 17.013 ng/µL and the concentration of tube 7 was 5.901 ng/µL.

Ligation of the restricted DNA

Following Ligation were performed at 37 °C for 60 minutes.

Tube		1	4	6	L7	L12	L13	L15	L16	L19
Product		pSB1C3-ldhA	pSB1C3-BBa_B0034-ldhA	pSB1C3-BBa_K20600-BBa_B0031-ldhA	pSB1C3-BBa_K20600-BBa_B0032-ldhA	pSB1C3-BBa_E0840-BBa_J45503	pSB1C3-cspD	pSB1C3-accA	pSB1C3-accB	pSB1C3-cbtA

Vector	Vector	pSB1C3-I20270	pSB1C3-I20270	pSB133-BBa_K206000	pSB133-BBa_K206000	pSB1C3-BBa_E0840	pSB1C3-I20270	pSB1C3-I20270	pSB1C3-I20270	pSB1C3-I20270
	Tube	5	5	4	4	7	5	5	5	5
	ρ	50 ng/µL	50 ng/µL	20 ng/µL	20 ng/µL	10 ng/µL	50 ng/µL	50 ng/µL	50 ng/µL	50 ng/µL
	Länge	2989 bp	2989 bp	2150 bp	2150 bp	2948 bp	2989 bp	2989 bp	2989 bp	2989 bp
	V	0.44 µL	1.05 µL	2.20 µL	1.46 µL	3.68 µL	0.74 µL	0.74 µL	0.73 µL	0.83 µL
Part 1	Part 1	ldhA	BBa_B0034-ldhA	BBa_B0031-ldhA	BBa_B0032-ldhA	BBa_J45503	cspD	accA	accB	cbtA
	Tube	15	18	16	17	9	10	11	12	13
	ρ	13 ng/µL	22 ng/µL	20 ng/µL	20 ng/µL	20 ng/µL	20 ng/µL	10 ng/µL	20 ng/µL	15 ng/µL
	Länge	1050 bp	273 bp	273 bp	522 bp	507 bp	507 bp	507 bp	522 bp	426 bp
	V	6.19 µL	2.17 µL	2.76 µL	3.58 µL	3.16 µL	3.15 µL	6.29 µL	3.18 µL	3.92 µL
Part 2	Part 2
	Tube
	ρ
	Länge
	V
T4 Buffer		1.00 µL	1.00 µL	1.00 µL	1.00 µL	1.00 µL	1.00 µL	1.00 µL	1.00 µL	1.00 µL
T4 Ligase		0.50 µL	0.50 µL	0.50 µL	0.50 µL	0.50 µL	0.50 µL	0.50 µL	0.50 µL	0.50 µL
H2O		1.87 µL	5.28 µL	3.54 µL	3.46 µL	1.66 µL	4.61 µL	1.47 µL	4.59 µL	3.76 µL
ad		10.00 µL	10.00 µL	10.00 µL	10.00 µL	10.00 µL	10.00 µL	10.00 µL	10.00 µL	10.00 µL

PCR of the 2nd Generation from e. coli and test restriction

All PCR Reactions were prepared as written below.

19 µL	H₂O
1 µL	DH5α
2.5 µL	Fw-Primer
2.5 µL	Rv-Primer
25 µL	Q5 High-Fidelity 2X Master Mix

Tube	Part	Fw-Primer	Rv-Primer	Predicted length (bp)	Concentration (ng/µL)
1	ldhA	Pre-ldhA-fw	ldhA-Suf-rv	1036	34.465
2	BBa_B0034-ldhA	34-ldhA-fw	ldhA-Suf-rv	1054	43.920
3	BBa_B0031-ldhA	31-ldhA-fw	ldhA-Suf-rv	1056	40.609
4	BBa_B0032-ldhA	32-ldhA-fw	ldhA-Suf-rv	1055	39.545
5	BBa_J45503	Pre-45503-fw	45503-Suf-rv	446	41.660
6	cspD	Pre-cspD-fw	cspD-Suf-rv	273	46.327
7	accA	Pre-accA-fw	accA-Suf-rv	1011	35.789
8	accB	Pre-accB-fw	accB-Suf-rv	522	X
9	tesA	Pre-tesA-fw	tesA-Suf-rv	678	38.747
10	cbtA	Pre-cbtA-fw	cbtA-Suf-rv	426	50.447
11	mraZ	Pre-mraZ-fw	mraZ-Suf-rv	507	X
12	LeuABCD	Pre-LeuA-fw	LeuD-Suf-rv	4748	X
13	LeuABCD6H	Pre-LeuA-fw	LeuD6H-Suf-rv	4766	X
14	OmpT	Pre-OmpT-fw	OmpT-Suf-rv	1002	X
15	MlcRE	Pre-MlcRE-fw	MlcRE-Suf-rv	252	54.880

PCR was performed using following Cycler-Protocol

Temperature (°C)	Time	Time (for LeuABCD)	Repeats
98	2:00	2:00	1
98	0:10	0:10	30
60	0:30	0:30	30
72	1:00	4:00	30
72	2:00	5:00	1
4	∞	∞	1

A 1% Agarose gel was prepared for gel electrophoresis.

The test restrction in the picture above was set up to check the activity of our restriction enzymes. The reactions were each set up with 11.5 µL H₂O, 6 µL of pSB1C3-I20270, 2 µL of NEB 2.1 buffer and 0.5 µL of restriction enzyme. Each tube was incubated at 37 °C for an hour and heat inacticated at 80 °C for 20 minutes.

Repetition of the restriction and the ligation of the 14^th June

Restriction and ligation were performed by pipetting following tables. Restriction was performed at 37 °C for 1 hour and heat inactivated for 20 minutes at 80 °C.

Tube

2

4

5

7

9

10

11

12

13

14

16

17

18

Part

pSB1C3-BBa_B0032

pSB133-BBa_K206000

pSB1C3-I20270

pSB1C3-BBa_E0840

BBa_J45503

cspD

accA

accB

cbtA

mraZ

BBa_B0031-ldhA

BBa_B0032-ldhA

BBa_B0034-ldhA

Lot#

180514NB07

180321NB01

180509NF01

180315NB01

aus PCR

ρ

81 ng/µL

140 ng/µL

166 ng/µL

128 ng/µL

42 ng/µL

46 ng/µL

30 ng/µL

36 ng/µL

39 ng/µL

50 ng/µL

41 ng/µL

40 ng/µL

44 ng/µL

Use

target m

200 ng

500 ng

200 ng

100 ng

200 ng

150 ng

300 ng

200 ng

Volumes

DNA

2.48 µL

1.43 µL

6.02 µL

0.78 µL

4.80 µL

4.32 µL

3.39 µL

5.59 µL

3.87 µL

5.95 µL

10.00 µL

NEBuffer 2.1

1.00 µL

2.00 µL

1.00 µL

2.00 µL

EcoRI-HF

0.50 µL

XbaI

0.50 µL

SpeI

0.50 µL

PstI

0.50 µL

FastAP

0.50 µL

H2O

5.02 µL

6.07 µL

10.48 µL

6.72 µL

3.20 µL

3.68 µL

4.61 µL

2.41 µL

4.13 µL

2.05 µL

7.00 µL

ad

10.00 µL

20.00 µL

10.00 µL

20.00 µL

Länge Produkt

ρ final

5.205 ng/µL

22.086 ng/µL

39.7 ng/µL

3.07 ng/µL

20 ng/µL

10 ng/µL

20 ng/µL

15 ng/µL

30 ng/µL

20 ng/µL

22 ng/µL

Gel extraction with the Thermo Scientific gel extraction kit was performed for tube 2, 4, 5 and 7

Ligation was performed over the weekend at 4 °C.

Tube

4

5

6

L7

L8

L12

L13

L15

L16

L17

L19

L21

L22

Product

pSB1C3-BBa_B0034-ldhA

pSB1C3-BBa_K20600-BBa_B0031-ldhA

pSB1C3-BBa_K20600-BBa_B0032-ldhA

pSB133-BBa_K206000-BBa_B0034-ldhA

pSB1C3-BBa_E0840-BBa_J45503

pSB1C3-cspD

pSB1C3-accA

pSB1C3-accB

pSB1C3-BBa_B0032-accB

pSB1C3-cbtA

pSB1C3-BBa_B0032-mraZ

pSB1C3-mraZ

Vector

Vector

pSB1C3-I20270

pSB133-BBa_K206000

pSB1C3-BBa_E0840

pSB1C3-I20270

pSB1C3-BBa_B0032

pSB1C3-I20270

pSB1C3-BBa_B0032

pSB1C3-I20270

Tube

5

4

7

5

2

5

2

5

ρ

50 ng/µL

20 ng/µL

10 ng/µL

50 ng/µL

20 ng/µL

50 ng/µL

20 ng/µL

50 ng/µL

Länge

2989 bp

2150 bp

2948 bp

2989 bp

2082 bp

2989 bp

2082 bp

2989 bp

V

1.05 µL

2.20 µL

1.46 µL

1.68 µL

3.68 µL

0.74 µL

0.73 µL

1.43 µL

0.83 µL

1.46 µL

0.74 µL

Part 1

Part 1

BBa_B0034-ldhA

BBa_B0031-ldhA

BBa_B0032-ldhA

BBa_B0034-ldhA

BBa_J45503

cspD

accA

accB

cbtA

mraZ

Tube

18

16

17

18

9

10

11

12

13

14

ρ

22 ng/µL

20 ng/µL

22 ng/µL

20 ng/µL

10 ng/µL

20 ng/µL

15 ng/µL

30 ng/µL

Länge

273 bp

522 bp

426 bp

507 bp

522 bp

426 bp

507 bp

V

2.17 µL

2.76 µL

3.58 µL

3.03 µL

3.16 µL

3.15 µL

6.29 µL

3.18 µL

3.57 µL

3.92 µL

2.36 µL

2.10 µL

T4 Buffer

1.00 µL

T4 Ligase

0.50 µL

H2O

5.28 µL

3.54 µL

3.46 µL

3.80 µL

1.66 µL

4.61 µL

1.47 µL

4.59 µL

3.50 µL

3.76 µL

4.68 µL

5.66 µL

ad

10.00 µL

PCR parts Generation 2 (missing parts)

Mastermix:

10 µL HF buffer
1 µL dNTPs
0.5 µL Phusion Polymerase
1 µL DNA
1.5 µL DMSO
1 µL fw primer
1 µL rv Primer
34 µL H₂O_dest

Tube	Part	Fw primer	Rv primer	Length	DNA concentration [ng/µL]
1	BBa_B0031-ldhA	31-ldhA-fw	ldhA-suf-rv	1056	44.465
2	BBa_345503	Pre-45503-fw	45503-suf-rv	446	44.646
3	tesA	Pre-tesA-fw	tesA-suf-rv	678	-
4	leuABCD	Pre-leuA-fw	leuD-suf-rv	4748	-
5	leuABCD6H	Pre-leuA-fw	leuD6H-suf-rv	4766	-
6	ompT	Pre-ompT-fw	ompT-suf-rv	1002	-

PCR program:

BBa_B0031_ldhA 1.1
BBa_345503 1.2
98°C	2 min
98°C	10 s
60°C	30 s
72°C	1 min
Go to step 2 30x
72°C	2 min

tesA 2.1
ompT 2.2
98°C	2 min
98°C	10 s
55°C	30 s
72°C	1 min
Go to step 2 25x
72°C	2 min

leuABCD 3.1
leuABCD6H 3.2
98°C	2 min
98°C	10 s
55°C	30 s
72°C	4 min
Go to step 2 30x
72°C	5 min

leuABCD with Phusion 4.1 and DMSO
leuABCD6H 4.2 and DMSO
98°C	3 min
98°C	10 s
60°C	30 s
72°C	4 min
Go to step 2 30x
72°C	5 min

PCR: leuABCD, ompT, tesA, psB1C3-Backbone and PCR Purification

PCR-Mastermix for leuABCD, ompT, tesA

Q5-Mastermix	10 µL
Primer fw	0.5 µL
Primer rev	0.5 µL
Template	0.5 µL
H₂0 ad 20 µL	8.5 µL

PCR-Mastermix for pSB1C3-Backbone

Q5-Mastermix	25 µL
Primer fw SB-prep-3p	1 µL
Primer rev SB-prep 2Ea	1 µL
Template	0.3 µL
H₂0 ad 50 µL	22.7 µL

Tube No.	Part
1	leuABCD
2	leuABCD-H
3	ompT
4	tesA
5	pSB1C3

PCR-program Tube 1.2

Step No.	Temperature	Time
1	98 ° C	2:00
2	98 ° C	0:10
3	50 ° C	0:30
4	72 ° C	3:00
5	Go to step 2 (30X)
6	72 ° C	2:00
7	4 ° C	∞

PCR-program Tube 3.4,5

Step No.	Temperature	Time
1	98 ° C	2:00
2	98 ° C	0:10
3	50 ° C	0:30
4	72 ° C	3:00
5	Go to step 2 (30X)
6	72 ° C	2:00
7	4 ° C	∞

Gel electrophoresis was performed using a 1% agarose gel

After gel electrophoresis only lane 5 (psB1C3) showed a band.

psB1C3 PCR product was purified with the Gene-Jet PCR-Purification kit.

Part	Lot No.	DNA conc
psB1C3-Backbone	180628NB01	96.6 ng/µL

Restriction and ligation of multiple Parts

Restriction Table

Tube

1

2

4

7

9

10

11

12

13

14

15

16

17

18

1

25

Part

pSB1C3-Bba_B0031

pSB1C3-Bba_B0032

pSB1C3-Bba_K206000

pSB1C3-Bba_E0840

Bba_J45503

cspD

accA

accB

cbtA

mreZ

ldhA

Bba_B0031-ldhA

Bba_B0032_ldhA

Bba_B0034_ldhA

pSB1C3

Lot#

180514NB01

180514NB07

180321NB01

180315NB01

aus PCR

180628NB01

ρ

153 ng/µL

81 ng/µL

47 ng/µL

128 ng/µL

42 ng/µL

44 ng/µL

30 ng/µL

36 ng/µL

39 ng/µL

50 ng/µL

34 ng/µL

41 ng/µL

40 ng/µL

97 ng/µL

Use

target m

200 ng

100 ng

200 ng

100 ng

200 ng

150 ng

300 ng

250 ng

100 ng

DNA

1.31 µL

1.24 µL

4.26 µL

0.78 µL

4.80 µL

4.32 µL

3.39 µL

5.59 µL

3.87 µL

5.95 µL

6.00 µL

4.00 µL

1.04 µL

NEBuffer 2.1

1.00 µL

EcoRI-HF

0.50 µL

XbaI

0.50 µL

SpeI

0.50 µL

PstI

0.50 µL

FastAP

0.50 µL

H₂O

6.19 µL

6.26 µL

3.24 µL

6.72 µL

3.20 µL

3.68 µL

4.61 µL

2.41 µL

4.13 µL

2.05 µL

2.00 µL

4.00 µL

6.96 µL

ad

10.00 µL

Länge Produkt

ρ final

20 ng/µL

10 ng/µL

20 ng/µL

10 ng/µL

20 ng/µL

10 ng/µL

20 ng/µL

15 ng/µL

30 ng/µL

21 ng/µL

16 ng/µL

18 ng/µL

10 ng/µL

Sequencing results from the 26^th of august 2018 showed that EcoR1 was falsely added to restriction No. 10.

Ligation Table

Tube

L1

L2

L3

L4

L5

L6

L7

L8

L9

L10

L11

L12

L13

L14

Product

pSB1C3-ldhA

pSB1C3-Bba_B0031-ldhA

pSB1C3-Bba_K206000-Bba_B0031-ldhA

pSB1C3-Bba_K206000-Bba_B0032_ldhA

pSB1C3-Bba_K206000-Bba_B0034_ldhA

pSB1C3-Bba_J45503

pSB1C3-Bba_E0840-Bba_J45503

pSB1C3-cspD

pSB1C3-Bba_B0031-cspD

pSB1C3-accA

pSB1C3-Bba_B0032-accB

pSB1C3-Bba_B0031-cbtA

pSB1C3-Bba_B0031-mraZ

pSB1C3-Bba_B0032-mraZ

Vector

Vector

pSB1C3

pSB1C3-Bba_B0031

pSB1C3-Bba_K206000

pSB1C3

pSB1C3-Bba_E0840

pSB1C3

pSB1C3-Bba_B0031

pSB1C3

pSB1C3-Bba_B0032

pSB1C3-Bba_B0031

pSB1C3-Bba_B0032

Tube

24

1

4

25

7

24

1

24

2

1

2

ρ

10 ng/µL

20 ng/µL

10 ng/µL

20 ng/µL

10 ng/µL

20 ng/µL

10 ng/µL

Länge

2989 bp

2081 bp

2150 bp

2989 bp

2948 bp

2070 bp

2081 bp

2989 bp

2082 bp

2081 bp

2082 bp

V

2.22 µL

1.59 µL

2.20 µL

1.46 µL

1.68 µL

4.12 µL

3.68 µL

4.27 µL

1.46 µL

3.71 µL

2.85 µL

1.64 µL

1.46 µL

2.91 µL

Part 1

Part 1

ldhA

Bba_B0031-ldhA

Bba_B0032_ldhA

Bba_B0034_ldhA

Bba_J45503

cspD

accA

accB

cbtA

mraZ

Tube

15

16

17

18

9

10

11

12

13

14

ρ

21 ng/µL

16 ng/µL

18 ng/µL

20 ng/µL

10 ng/µL

20 ng/µL

15 ng/µL

30 ng/µL

Länge

1050 bp

446 bp

273 bp

426 bp

507 bp

273 bp

507 bp

522 bp

426 bp

507 bp

V

3.76 µL

3.30 µL

3.44 µL

4.48 µL

3.78 µL

2.94 µL

3.16 µL

2.84 µL

3.54 µL

6.29 µL

3.57 µL

4.48 µL

2.36 µL

Part 2

Part 2

Tube

ρ

Länge

V

T4 Buffer

1.00 µL

2.00 µL

1.00 µL

T4 Ligase

0.50 µL

H2O

2.52 µL

3.61 µL

2.85 µL

2.56 µL

1.44 µL

1.38 µL

3.50 µL

7.50 µL

2.08 µL

2.38 µL

4.68 µL

3.23 µL

ad

10.00 µL

20.00 µL

10.00 µL

Restriction and ligation of multiple Parts

Restriction Table

Tube

1

2

4

7

9

10

11

12

13

14

15

16

17

18

1

25

Part

pSB1C3-Bba_B0031

pSB1C3-Bba_B0032

pSB1C3-Bba_K206000

pSB1C3-Bba_E0840

Bba_J45503

cspD

accA

accB

cbtA

mreZ

ldhA

Bba_B0031-ldhA

Bba_B0032_ldhA

Bba_B0034_ldhA

pSB1C3

Lot#

180514NB01

180514NB07

180321NB01

180315NB01

aus PCR

180628NB01

ρ

153 ng/µL

81 ng/µL

47 ng/µL

128 ng/µL

42 ng/µL

44 ng/µL

30 ng/µL

36 ng/µL

39 ng/µL

50 ng/µL

34 ng/µL

41 ng/µL

40 ng/µL

97 ng/µL

Use

target m

200 ng

100 ng

200 ng

100 ng

200 ng

150 ng

300 ng

250 ng

100 ng

DNA

1.31 µL

1.24 µL

4.26 µL

0.78 µL

4.80 µL

4.32 µL

3.39 µL

5.59 µL

3.87 µL

5.95 µL

6.00 µL

4.00 µL

1.04 µL

NEBuffer 2.1

1.00 µL

EcoRI-HF

0.50 µL

XbaI

0.50 µL

SpeI

0.50 µL

PstI

0.50 µL

FastAP

0.50 µL

H₂O

6.19 µL

6.26 µL

3.24 µL

6.72 µL

3.20 µL

3.68 µL

4.61 µL

2.41 µL

4.13 µL

2.05 µL

2.00 µL

4.00 µL

6.96 µL

ad

10.00 µL

Länge Produkt

ρ final

20 ng/µL

10 ng/µL

20 ng/µL

10 ng/µL

20 ng/µL

10 ng/µL

20 ng/µL

15 ng/µL

30 ng/µL

21 ng/µL

16 ng/µL

18 ng/µL

10 ng/µL

Sequencing results from the 26^th of august 2018 showed that EcoR1 was falsely added to restriction No. 10.

Ligation Table

Tube

L1

L2

L3

L4

L5

L6

L7

L8

L9

L10

L11

L12

L13

L14

Product

pSB1C3-ldhA

pSB1C3-Bba_B0031-ldhA

pSB1C3-Bba_K206000-Bba_B0031-ldhA

pSB1C3-Bba_K206000-Bba_B0032_ldhA

pSB1C3-Bba_K206000-Bba_B0034_ldhA

pSB1C3-Bba_J45503

pSB1C3-Bba_E0840-Bba_J45503

pSB1C3-cspD

pSB1C3-Bba_B0031-cspD

pSB1C3-accA

pSB1C3-Bba_B0032-accB

pSB1C3-Bba_B0031-cbtA

pSB1C3-Bba_B0031-mraZ

pSB1C3-Bba_B0032-mraZ

Vector

Vector

pSB1C3

pSB1C3-Bba_B0031

pSB1C3-Bba_K206000

pSB1C3

pSB1C3-Bba_E0840

pSB1C3

pSB1C3-Bba_B0031

pSB1C3

pSB1C3-Bba_B0032

pSB1C3-Bba_B0031

pSB1C3-Bba_B0032

Tube

24

1

4

25

7

24

1

24

2

1

2

ρ

10 ng/µL

20 ng/µL

10 ng/µL

20 ng/µL

10 ng/µL

20 ng/µL

10 ng/µL

Länge

2989 bp

2081 bp

2150 bp

2989 bp

2948 bp

2070 bp

2081 bp

2989 bp

2082 bp

2081 bp

2082 bp

V

2.22 µL

1.59 µL

2.20 µL

1.46 µL

1.68 µL

4.12 µL

3.68 µL

4.27 µL

1.46 µL

3.71 µL

2.85 µL

1.64 µL

1.46 µL

2.91 µL

Part 1

Part 1

ldhA

Bba_B0031-ldhA

Bba_B0032_ldhA

Bba_B0034_ldhA

Bba_J45503

cspD

accA

accB

cbtA

mraZ

Tube

15

16

17

18

9

10

11

12

13

14

ρ

21 ng/µL

16 ng/µL

18 ng/µL

20 ng/µL

10 ng/µL

20 ng/µL

15 ng/µL

30 ng/µL

Länge

1050 bp

446 bp

273 bp

426 bp

507 bp

273 bp

507 bp

522 bp

426 bp

507 bp

V

3.76 µL

3.30 µL

3.44 µL

4.48 µL

3.78 µL

2.94 µL

3.16 µL

2.84 µL

3.54 µL

6.29 µL

3.57 µL

4.48 µL

2.36 µL

Part 2

Part 2

Tube

ρ

Länge

V

T4 Buffer

1.00 µL

2.00 µL

1.00 µL

T4 Ligase

0.50 µL

H2O

2.52 µL

3.61 µL

2.85 µL

2.56 µL

1.44 µL

1.38 µL

3.50 µL

7.50 µL

2.08 µL

2.38 µL

4.68 µL

3.23 µL

ad

10.00 µL

20.00 µL

10.00 µL

Restriction and Ligation of PCR Gen II and Transformation

Restriction:

Tube

1

2

3

4

5

6

7

8

9

10

11

12

13

14

15

16

17

18

19

20

21

22

23

24

25

Part

pSB1C3-BBa_B0030

pSB1C3-BBa_B0031

pSB1C3-BBa_B0032

pSB1C3-BBa_B0031

pSB133-BBa_K206000

pSB1C3-I20270

pSB1C3-BBa_E0840

pSB1C3-I20270

BBa_J45503

cspD

accA

accB

cbtA

mraZ

ldhA

BBa_B0031-ldhA

BBa_B0032-ldhA

BBa_B0034-ldhA

MlcRE

ldhA

cspD

accB

mraZ

pSB1C3

Lot#

180514NB01

180514NB07

180514NB01

180321NB01

180509NF01

180315NB01

180509NF01

aus PCR

180628NB01

ρ

153 ng/µL

81 ng/µL

153 ng/µL

47 ng/µL

166 ng/µL

128 ng/µL

166 ng/µL

42 ng/µL

46 ng/µL

30 ng/µL

36 ng/µL

39 ng/µL

50 ng/µL

34 ng/µL

41 ng/µL

40 ng/µL

44 ng/µL

55 ng/µL

34 ng/µL

46 ng/µL

36 ng/µL

50 ng/µL

97 ng/µL

Use

target m

200 ng

100 ng

200 ng

500 ng

200 ng

100 ng

200 ng

150 ng

300 ng

250 ng

100 ng

250 ng

200 ng

300 ng

DNA

1.31 µL

1.24 µL

1.31 µL

4.26 µL

6.02 µL

0.60 µL

0.78 µL

0.60 µL

4.80 µL

4.32 µL

3.39 µL

5.59 µL

3.87 µL

5.95 µL

6.00 µL

4.00 µL

2.00 µL

7.30 µL

4.32 µL

5.59 µL

5.95 µL

1.04 µL

NEBuffer 2.1

1.00 µL

2.00 µL

1.00 µL

2.00 µL

1.00 µL

EcoRI-HF

0.50 µL

XbaI

0.50 µL

SpeI

0.50 µL

PstI

0.50 µL

FastAP

0.50 µL

H₂O

7.50 µL

6.19 µL

6.26 µL

6.19 µL

3.24 µL

10.48 µL

6.90 µL

6.72 µL

6.90 µL

3.20 µL

3.68 µL

4.61 µL

2.41 µL

4.13 µL

2.05 µL

2.00 µL

4.00 µL

6.00 µL

9.70 µL

3.68 µL

2.41 µL

2.05 µL

6.96 µL

ad

10.00 µL

20.00 µL

10.00 µL

20.00 µL

10.00 µL

Länge Produkt

ρ final

0 ng/µL

20 ng/µL

10 ng/µL

20 ng/µL

50 ng/µL

10 ng/µL

20 ng/µL

10 ng/µL

20 ng/µL

15 ng/µL

30 ng/µL

21 ng/µL

16 ng/µL

18 ng/µL

11 ng/µL

13 ng/µL

20 ng/µL

30 ng/µL

10 ng/µL

24

15

13 ng/µL

10

15

13 ng/µL

10

15

The parts were restricted at 37°C for 1 h and after that stored at 80°C and 20 min.

Ligation:

pSB1C3-ldhA

pSB1C3-BBa_B0031-ldhA

pSB1C3-BBa_B0032-ldhA

pSB1C3-BBa_B0034-ldhA

pSB1C3-BBa_K20600-BBa_B0031-ldhA

pSB1C3-BBa_K20600-BBa_B0032-ldhA

pSB133-BBa_K206000-BBa_B0034-ldhA

pSB1C3-BBa_J45503

pSB1C3-BBa_E0840-BBa_J45503

pSB1C3-cspD

pSB1C3-BBa_B0031-cspD

pSB1C3-accA

pSB1C3-accB

pSB1C3-BBa_B0032-accB

pSB1C3-BBa_B0031-cbtA

pSB1C3-cbtA

pSB1C3-BBa_B0031-mraZ

pSB1C3-BBa_B0032-mraZ

pSB1C3-mraZ

pSB1C3-MlcRE

pSB1C3-MlcRE-BBa_B0032-ldhA

pSB1C3-MlcRE-BBa_B0034-ldhA

pSB1C3-BBa_B0030-accA

pSB1C3

pSB1C3-BBa_B0031

pSB1C3-BBa_B0032

pSB1C3-I20270

pSB133-BBa_K206000

pSB1C3

pSB1C3-BBa_E0840

pSB1C3

pSB1C3-BBa_B0031

pSB1C3

pSB1C3-BBa_B0032

pSB1C3-BBa_B0031

pSB1C3

pSB1C3-BBa_B0031

pSB1C3-BBa_B0032

pSB1C3

pSB1C3-I20270

pSB1C3-BBa_B0030

24

1

2

5

4

25

7

24

1

24

2

1

24

1

2

24

6

10 ng/µL

20 ng/µL

10 ng/µL

50 ng/µL

20 ng/µL

10 ng/µL

20 ng/µL

10 ng/µL

20 ng/µL

10 ng/µL

20 ng/µL

10 ng/µL

2989 bp

2081 bp

2084 bp

2989 bp

2150 bp

2989 bp

2948 bp

2989 bp

2081 bp

2989 bp

2082 bp

2081 bp

2989 bp

2081 bp

2082 bp

2989 bp

2085 bp

2.22 µL

1.59 µL

4.33 µL

1.05 µL

2.20 µL

1.46 µL

1.68 µL

4.12 µL

3.68 µL

3.71 µL

1.46 µL

3.71 µL

3.64 µL

2.85 µL

1.64 µL

4.12 µL

1.46 µL

2.91 µL

3.71 µL

1.86 µL

ldhA

BBa_B0034-ldhA

BBa_B0031-ldhA

BBa_B0032-ldhA

BBa_B0034-ldhA

BBa_J45503

cspD

accA

accB

cbtA

mraZ

MlcRE

15

18

16

17

18

9

10

11

12

13

14

19

21 ng/µL

18 ng/µL

16 ng/µL

18 ng/µL

20 ng/µL

10 ng/µL

20 ng/µL

15 ng/µL

30 ng/µL

11 ng/µL

1050 bp

446 bp

273 bp

522 bp

426 bp

507 bp

522 bp

426 bp

507 bp

252 bp

3.76 µL

3.30 µL

2.74 µL

2.72 µL

3.44 µL

4.48 µL

3.78 µL

2.94 µL

3.16 µL

3.15 µL

3.54 µL

6.29 µL

3.18 µL

3.57 µL

4.48 µL

3.92 µL

2.36 µL

2.10 µL

1.43 µL

BBa_B0032-ldhA

BBa_B0034-ldhA

accA

16

17

18

16 ng/µL

18 ng/µL

1054 bp

1055 bp

1056 bp

507 bp

4.48 µL

4.60 µL

4.14 µL

1.00 µL

2.00 µL

1.00 µL

0.50 µL

2.52 µL

3.61 µL

1.43 µL

4.74 µL

2.85 µL

2.56 µL

3.04 µL

1.44 µL

1.66 µL

1.65 µL

3.50 µL

7.50 µL

1.68 µL

2.07 µL

2.38 µL

0.46 µL

4.68 µL

3.23 µL

2.69 µL

0.73 µL

0.61 µL

1.07 µL

10.00 µL

20.00 µL

10.00 µL

The parts were ligated at 37°C for 1 h.

Colony-PCR (Gen.II Parts)

1-3	180629NiF01	pSB1C3-ldhA	X
4-6	180629NiF02	pSB1C3-BBa_B0031-ldhA	✓6
7-9	180629NiF03	pSB1C3-BBa_K206000-BBa_B0031-ldhA	X
10-12	180629NiF04	pSB1C3-BBa_K206000-BBa_B0032-ldhA	✓11-13
13-15	180629NiF05	pSB1C3-BBa_K206000-BBa_B0034-ldhA	✓15
16-18	180629NiF06	pSB1C3-BBa_J45503	X
19-21	180629NiF07	pSB1C3-BBa_E0840-BBa_J45503	✓20
22-24	180629NiF08	pSB1C3-cspD	✓22
25-27	180629NiF09	pSB1C3-BBa_B0031-cspD	X
28-30	180629NiF10	pSB1C3-accA	X
31-33	180629NiF11	pSB1C3-BBa_B0032-accB	✓32
34-36	180629NiF12	pSB1C3- BBa_B0031-cbtA	X
37-39	180629NiF13	pSB1C3- BBa_B0031-mraz	X
40-42	180629NiF14	pSB1C3- BBa_B0032-mraz	✓40-42

For 45 Uses

PCR-Program

95 °C	3 min
95 °C	30 s
55 °C	30 s
72 °C	1:30 min
72 °C	5 min

Lot-Numbers

6 à 180702NB01: psB1C3-BBa_B0031-ldhA

11 à 180702NB02: psB1C3-BBa_K206000-BBa-B0032-ldhA

12 à 180702NB03: psB1C3-BBa_K206000-BBa-B0032-ldhA

13 à 180702NB04: psB1C3-BBa_K206000-BBa-B0032-ldhA

15 à 180702NB05: psB1C3-BBa_ K206000-BBa-B0034-ldhA

20 à 180702NB06: psB1C3-BBa_E0840-JU5503

22 à 180702NB07 psB1C3-cspD

32 à 180702NB08: psB1C3-BBa_B0032-accB

40 à 180702NB09: psB1C3-BBa_B0032-mraz

41 à 180702NB10: psB1C3-BBa_B0032-mraz

42 à 180702NB11: psB1C3-BBa_B0032-mraz

https://static.igem.org/mediawiki/2018/7/7a/T--Hamburg--180702_cPCR1.JPG

Colony-PCR (Gen.II Parts)

1-3	180629NiF01	pSB1C3-ldhA	X
4-6	180629NiF02	pSB1C3-BBa_B0031-ldhA	✓6
7-9	180629NiF03	pSB1C3-BBa_K206000-BBa_B0031-ldhA	X
10-12	180629NiF04	pSB1C3-BBa_K206000-BBa_B0032-ldhA	✓11-13
13-15	180629NiF05	pSB1C3-BBa_K206000-BBa_B0034-ldhA	✓15
16-18	180629NiF06	pSB1C3-BBa_J45503	X
19-21	180629NiF07	pSB1C3-BBa_E0840-BBa_J45503	✓20
22-24	180629NiF08	pSB1C3-cspD	✓22
25-27	180629NiF09	pSB1C3-BBa_B0031-cspD	X
28-30	180629NiF10	pSB1C3-accA	X
31-33	180629NiF11	pSB1C3-BBa_B0032-accB	✓32
34-36	180629NiF12	pSB1C3- BBa_B0031-cbtA	X
37-39	180629NiF13	pSB1C3- BBa_B0031-mraz	X
40-42	180629NiF14	pSB1C3- BBa_B0032-mraz	✓40-42

For 45 Uses

PCR-Program

95 °C	3 min
95 °C	30 s
55 °C	30 s
72 °C	1:30 min
72 °C	5 min

https://static.igem.org/mediawiki/2018/5/59/T--Hamburg--180702_cPCR2.JPG

180704 – 1

oHybB characterisation

Cultures for characterisation were set up at an OD of 0.1 employing over night cultures from the day before.

Cultures were incubated for 6 hours at different temperatures. The optical density was measured each hour. At each time point 1 mL of culture was taken and added to 600 µL of PFA and stored in the fridge until measurement.

	18 °C				27 °C				37 °C
t [h]	DH5α (-)	I20270 (+)	oHybB	J45503	DH5α (-)	I20270 (+)	oHybB	J45503	DH5α (-)	I20270 (+)	oHybB	J45503
0	0.134	0.074	0.100	0.102	0.140	0.068	0.102	0.108	0.098	0.082	0.194	0.110
1	0.252	0.060	0.098	0.098	0.196	0.084	0.170	0.170	0.430	0.260	0.490	0.340
2	0.210	0.088	0.144	0.160	0.836	0.400	0.924	0.788	1.376	0.756	1.448	0.980
3	0.304	0.112	0.208	0.210	0.952	0.372	0.920	0.784	3.120	2.008	3.544	2.352
4	0.440	0.164	0.312	0.282	1.792	0.832	1.624	1.416	3.920	-	3.890	4.410
5	0.658	0.202	0.430	0.404	2.920	2.010	2.030	2.440	5.540	-	5.360	6.300
6	1.240	0.352	0.736	0.680	4.700	3.240	4.640	4.920	7.200	-	6.960	8.500

GFP measurement was carried out the next day.

Evaluation of oHybB-characterization

Plate-reader measured with following load-schematic:

18°C

27°C

37°C

1

2

3

4

5

6

7

8

9

10

11

12

t₁

A

-

+

oHybB

J45503

-

+

oHybB

J45503

-

+

oHybB

J45503

t₂

B

-

+

oHybB

J45503

-

+

oHybB

J45503

-

+

oHybB

J45503

t₃

C

-

+

oHybB

J45503

-

+

oHybB

J45503

-

+

oHybB

J45503

t₄

D

-

+

oHybB

J45503

-

+

oHybB

J45503

-

+

oHybB

J45503

t₅

E

-

+

oHybB

J45503

-

+

oHybB

J45503

-

+

oHybB

J45503

t₆

F

-

+

oHybB

J45503

-

+

oHybB

J45503

-

+

oHybB

J45503

- = DH5a

+ = pSB1C3-I20270

Load-schematic for all temperatures:

The rows G and H were added to the plates 27°C and 37°C as a reference. There was no positive control from 4 at 37°C. There was no positive and negative control with 37°C at t₆.

1

2

3

4

5

6

7

8

9

10

11

12

t₁

A

-

+

oHybB

J45503

t₂

B

-

+

oHybB

J45503

t₃

C

-

+

oHybB

J45503

t₄

D

-

+

oHybB

J45503

t₅

E

-

+

oHybB

J45503

t₆

F

-

+

oHybB

J45503

18°C

G

+ t_x

H

TD-PCR (PCR-GGA)

PCR of LeuABCD, LeuABCD6H and tesA was performed.

Two different master mixes were set up.

Mastermix1

Substances	1x volume (in µl)
Q5-master mix	2.5
F-Primer	2.5
R-Primer	2.5
DNA (DH5α)	1
DMSO	1.5
ddH₂O	7.5

Mastermix 2

Substances	1x volume (in µl)
10x Taq buffer	2
Taq Polymerase	0.5
F-Primer	1.2
R-Primer	1.2
DNA (DH5α)	1
dNTPs	0.4
50 mM MgCl₂	0.6
DMSO	1.5
ddH₂O	13

PCR-Program

95 °C	3 min
95 °C	30 s
60 °C	30 s
72 °C	1:30 min
72 °C	5 min

Start-Annealing was set to 60 °C. Every cycle the temperature was set down 2 °C until 40 °C was reached following 20 cycles at 40°C.

PCR Golden Gate Assembly (GGA)

14x PCR-set ups with 50 μl with Q5-Polymerase (NEB)

10 μM 2.5 μL fw-Primer, 10 μM 2.5 μL rv-Primer, 25 μL Q5 2x Master Mix

Template DNA: 0.1 μL Plasmid or 0.5 μL DH5α, 20 μL H₂O

Product	Fw-Primer	Rv-Primer	Pred. length	Template	End length
BsaI30cbtABsaI	BsaI30cbtA	cbtA_BsaI_r	394	cbtA
BsaI30sspDBsaI	BsaI30sspD	sspD_BsaI_r	244	sspD
BsaI30mrazBsaI	BsaI30mraz	Mraz_BsaI_r	408	mraZ
BsaI30SulABsaI	BsaISulA	SulA_Suf_Bsa	550	SulA
BsaI32cbtABsaI	BsaI32cbtA	cbtA_BsaI_r	394	cbtA
BsaI32sspDBsaI	BsaI32sspD	sspD_BsaI_r	244	sspD
BsaI32mrazBsaI	BsaI32mraz	Mraz_BsaI_r	408	mraZ
BsaI32SulABsaI	BsaISulA	SulA_Suf_Bsa	550	pSB1C3SulA
BsaI34cbtABsaI	BsaI34cbtA	cbtA_BsaI_r	394	cbtA
BsaI34sspDBsaI	BsaI34sspD	sspD_BsaI_r	244	sspD
BsaI34mrazBsaI	BsaI34mraz	Mraz_BsaI_r	408	mraZ
BsaI34SulABsaI	BsaISulA	SulA_Suf_Bsa	550	pSB1C3SulA
dapAP	dapAP_s_f	dapAP_s_r	120	synthetic
dapAP_BsaI	BsaI_dapAP_f	dapAP_s_r	120	DH5α

PCR-Progamm:

Temperature	Time
98 °C	0:30 (2:00 für DH5α)
98 °C	0:10
55 °C	0:20
72 °C	1:00 (Go to step 2, repeat 30 times)
72 °C	2:00

Tubes 1-14 à Lotnumber 180706BK01-14

Golden Gate Assembly (Restriction and Ligation)

Tube	H2O	GGABulter	GGAM	pGGA	180706BK…
1	14 µL	2 µL	1 µL	1 µL	0.15 µL (01-12)
2	14 µL	2 µL	1 µL	1 µL	0.15 µL (01-12)
3	14 µL	2 µL	1 µL	1 µL	0.8 µL (12), 0.55 µL (05), 0.4 µL (06), 0.43 µL (07)
4	14 µL	2 µL	1 µL	1 µL	0.55 µL (04), 0.55 µL (05), 0.4 µL (06), 0.43 µL (07)
5	14 µL	2 µL	1 µL	1 µL	0.8 µL (12), 0.4 µL (01), 0.3 µL (02), 0.3 µL (03)

GGA Program:

37°C	1 min
16°C	1 min
Go to step 1 30x
55°C	5 min

Transformation

Protocol by Zhang:

180710BK02 = Tube 1
180710BK03 = Tube 2
180710BK04 = Tube 3
180710BK05 = Tube 4
180710BK06 = Tube 5

180711 – 1

cPCR – of trafos from GGA of the growth inhibition with different ribosome binding sites and RNA G120

cPCR was performed as the next step of the transformations of the previous day. Colonies were picked and used for the cPCR as follows:

Plate No. of Colonies Tube No.

               180710NB02       4                            01 – 04
               180710BK02       9                            05 – 13
               180710BK03       14                          14 – 27
               180710BK04       4                            28 – 31
               180710BK05       4                            32 – 35
               180710BK06       4                            36 – 39

Master Mix was set up for 40 reactions with:

80 µL of 10x Taq Buffer
16 µL of 10 nM DNTPs
14 µL of 50 nM MgCl₂
2 µL of Taq DNA Polymerase
631 µL of H₂0

Forward and reversed primers were added with 0.6 µL each. For the GGA cPCR the primers used were GGA_cPCR_f (forward) and GGA_cPCR_r (reversed). For the cPCR for RNA G120 the primers used were Vf2 (forward) and Vr (reversed).

PCR was carried out according to the following protocol:

step T (°C) t (min)

1             95           3:00
2             95           0:20
3             56           0:30
4             72           2:30
5             72           5:00
6             6             hold

Cycle from step 2 to 4 was repeated 30 times.

Results:

cPCR of RNA-G120 operator worked and we got a part with the desired length.

cPCR of the colonies from the GGA colonies didn’t work well. Just one colonie (lane 32) had a part with the desired length (~ 2000 bp).

Restriktion, Ligation, Transformation, RNAG120-Operator

Restriktion:

	RNA-Operator 180709NB01	pSB1C3-E084018020BN03
c_DNA	144.037 ng/μL	57 ng/μL
V_DNA	0.69 μL	1.75 μL
NEB-Buffer	1 μL	1 μL
EcoRI	0.5 μL	0.5 μL
SpeI	0.5 μL
XbaI		0.5 μL
FastAP		0.5 μL
H₂O	7.31 μL	5.75 μL
Total	10 μL	10 μL

1 h, 37 °C + 20 min 80 °C heat shock deactivation

GGA Miniprep-Cryostock (rRNA G120) (check cPCR from 180711)

Culture 32: 180712BK01 Plasmid (c = 169.243 ng/μL)

180712BK02 Cryostock

Culture 2: 180712BK03 Plasmid (c = 93.328 ng/μL)

180712BK04 Cryostock

PCR

1 – kivD

2 – accC

3 – accD

4 – BjaII-OmpT-SiteF-A6

Approach:

20 μL ddH₂O

25 μL Q5-Mix

2.5 μL Fw-Primer

2.5 μL Rev-Primer

0.2 μL Template

Total of: 50.2 μL

Vectorrestriction:

	pSB1C3_BBa_320270	BBa_E0840	pSB1C3_RNA_G120
DNA	12.05 μL	6 μL	6 μL
NEB 2.1	2 μL	1 μL	1 μL
EcoRI	0.4 μL	0.2 μL	0.2 μL
PstI	0.4 μL	0.2 μL	0.2 μL (SpeI)
FastAP	0.5 μL	0.5 μL	0.5 μL
H₂O	4.65 μL	2.1 μL	2.1 μL

Run Samples from PCR and restriction on 1%-agarosegel:

Gelextraction

Actions performed according to ThermoFischers GeneJet GelEx-Kit.

DNA	concentration	Lot-Nr.
pSB1C3	30 ng/μL	180716LP01
pSB1C3_BBa_E0840	3.9 ng/μL	180716LP02
RNAG120-Operator-MIcRE	5.9 ng/μL	180716LP03
kivD	15.2 ng/μL	180716LP04
accC	10 ng/μL	180716LP05
accD	7.5 ng/μL	180716LP06
BjaII-OmpT-SiteF-A6	110 ng/μL	180716LP07

Ligation:

	L2	L4	L5	L6	L7
DNA	pSB1C3_E0840 (9.29 μL)	pSB1C3 (0.4 μL)	pSB1C3 (1.47 μL)	pSB1C3 (0.04 μL)	pSB1C3 (0.62 μL)
DNA	RNAG120 (10.81 μL)	kivD (6.7 μL)	BjaII-OmpT-SiteF-A6 (0.5 μL)	accC (8.67 μL)	accD (10.82 μL)
T4-Buffer	3 μL	1 μL	1 μL	2 μL	2 μL
T4-Ligase	0.5 μL	0.5 μL	0.5 μL	0.5 μL	0.5 μL
H₂O	6.4 μL	1.4 μL	6.54 μL	8.39 μL	6.02 μL

Incubate over-night at 16 °C

Characterisation of the growth inhibition module

Growth inhibition module: pGGA-BBa_B0032-cbtA-BBa_B0032-cspD-BBa_B0032-mraZ-BBa_B0030-sulA

Characterisation of the growth inhibition module with DH5α without the part as a negative control (DH5α (-)). Diaminopimelic acid (DAP) used for repression of the growth inhibition at a concentration of 40 µM and at high concentration of 8.3 mM (DAP*).

Initial OD measurements

O/n culture OD µL taken to get an OD of 0.01 in 20 µL media

               DH5α (-)               1.78       112.35
               DH5α                    1.23       162.60
               DH5α + DAP        1.18       169.49
               DH5α + DAP*      1.33       150.38

For the characterisation all cultures started with an OD of 0.01 in 20 mL.

For the characterisation the cultures were incubated at 37 °C for 6 hours, with a measurement of the OD at each full hour. The results were as follows:

t [h]	DH5α (-)	DH5α	DH5α + DAP	DH5α + DAP*
0	0.01	0.01	0.01	0.01
1	0.133	0.009	0.011	0.025
2	0.034	0.022	0.021	0.058
3	0.172	0.027	0.030	0.064
4	0.711	0.156	0.148	0.303
5	1.162	0.28	0.205	0.678
6	1.422	0.593	0.459	1.145

Results:

Dap-repressed and unrepressed samples showed similar growth behaviour. However, at high dap concentrations the cultures grew faster. Cells without growth-inhibiting plasmid grew best.

Colony PCR of the transformation from 17.07.18

A cPCR was performed of the transformation from the previous day. A PCR Mastermix was created:

Taq 10x Buffer	50 µL
dNTPs mM	10 µL
MgCl₂50mM	15 µL
Taq DNA Polymerase	1 µL
VF2-Primer	15 µL
VR-Primer	15 µL
H₂O	394 µL

Colonies were picked as follows:

Part	Tube No.
pSB1C3-BBa_R0010	1-4
pSB1C3-BBa_K577881	5-8
pSB1C3-BBa_E0240	9-12
pSB1C3-BBa_kivD	13-16
pSB1C3-BBa_BjaIT-OmpTFlag	17-20
pSB1C3-BBa_accd	21-24

180720

Miniprep and Cryostocks

Cryostocks were created by adding 200 µL 50% glycose to 600µL LB-Medium

Part	Lot No.
pSB1C3-BBa_R0010	180720BK01
pSB1C3-BBa_1577881	180720BK02

Minipreps were performed using the Thermo Fischer Kit

Part	Lot No.	Concentration [ng/µL]
pSB1C3-BBa_R00010	180720BK03	80.454
pSB1C3-BBa_1572881	180720BK04	188.945

PCR of synthetic Part

Approach: 20 μL H₂O, 25 μL Q5-Mastermix, 2.5 μL Syn_f-Primer, 2.5 μL Syn_r-Primer,

DNA-Template from Table:

Lot-Nr.	concentration	Part
180723BK01	171.027 ng/ μL	ADH2
180723BK02	117.758 ng/ μL	accC
180723BK03	115.077 ng/ μL	kivD
180723BK04	105.878 ng/ μL	OmpA-NnAOX1a

Purifictation with ThermoFischer-Kit

PCR-Programm:

Temperature	Time
98 °C	20 s
98 °C	10 s
55 °C	20 s (Go to Step 1, repeat 30 times)
72 °C	1:20 min
72 °C	2:00 min

Restriction of pSB1C3 (linear, PCR-product) + 1 % Agarosegel

180628NB01 à pSB1C3 DNA	15 μL
NEB 2.1	2 μL
EcoRI-HF	0.5 μL
PstI	0.5 μL
FastAP	0.5 μL
H₂O	1.5 μL

Incubate at 37 °C for 60 minutes, then at 80 °C for heat shock deactivation.

Interlab

Pecked each of 2 colonies and put in a 7 mL LB-cAMP and stored 1 h at 37°C and 200 rpm.

OD-Measure by 600 nm:

Tube	A1	1.1	1.2	1.3	A2	2.1	2.2	2.3
OD	0.2892	0.152	0.158	0.156	0.2216	0.153	0.153	0.152

Tube	B1	1.1	1.2	1.3	B2	2.1	2.2	2.3
OD	0.262	0.143	0.145	0.156	0.2936	0.174	0.183	0.173

Tube	C1	C2
OD	0.048	0.048

PCR for GGA and Over-night-cultures (OVC) for sequencing

Part	Fw-Primer	Rv-Primer	Template	Lotnumber	DNA-concentration [ng/µL]	Tube
accA	Bsa1_accA_f	Bsa1_accA_r	180622NB05	180730BK01	174.035	1
accB	Bsa1_accB_f	Bsa1_accB_r	180703BK05	180730BK02	92.21	2
accC	Bsa1_accC_f	Bsa1_accC_r	180726LP04	180730BK03	17.536	3
accD	Bsa1_accD_f	Bsa1_accD_r	IDT	180730BK04	189.443	4
tesA	Bsa1_tesA_f	Bsa1_tesA_r	e.coli	180730BK05	111.71	5
ADH2	Bsa1_ADH2_f	Bsa1_ADH2_r	180726LP02	180730BK06	62.293	6
hivD	Bsa1_hivD_f	Bsa1_hivD_r	180726LP01	180730BK07	91.148	7
LeuA	Bsa1_LeuA_f	Bsa1_LeuA_r	e.coli	180730BK08	88.001	8
LeuB	Bsa1_LeuB_f	Bsa1_LeuB_r	e.coli	180730BK09	61.115	9
LeuC	Bsa1_LeuC_f	Bsa1_LeuC_r	e.coli	180730BK10	93.537	10
LeuD	Bsa1_LeuD_f	Bsa1_LeuD_r	e.coli	180730BK11	109.97	11
BjaIT	Bsa1_BjaIT_f	Bsa1_BjaIT_r	IDT	180730BK12	127.138	12
HlyA	Bsa1_HlyA_f	Bsa1_HlyA_r	pSB1C3-K554002	180730BK13	39.76	13
pBAD	Bsa1_pBAD_f	Bsa1_pBAD_r	pSB1C3-K206000	180730BK14	99.724	14
HlyB	Bsa1_HlyB_f	Bsa1_HlyB_r	pSB1C3-K554013	180730BK15	205.409	15
OmpT	Bsa1_OmpT_f	Bsa1_OmpT_r	e.coli	180730BK16	48.8	16
TolC	Bsa1_TolC_f	Bsa1_TolC_r	pSB1C3-K554013	180730BK17	207.256	17
Exer.s	Bsa1_HlyB_f	Bsa1_TolC_r	pSB1C3-K554013	180730BK18	207.123	18

Mastermix:

20 µL H₂O_dest
5 µL each Primer (fw and rv)
3 µL template
25 µL Q5 Mastermix

PCR-Programm:

98°C	20 s
98°C	10 s
55°C	20 s
72°C	2 min
Go to step 2 30x
72°C	2 min

Mini-prep of the over-night-cultures (OVC) for the sequencing:

The mini-prep was implemented with GeneJet Plasmid Mini-prep kit. The DNA we eluted with H₂O_dest.

Lot-number	Part	DNA-concentration [ng/µL]
180731NF01	pSB1C3_MlcRE_BBa_E0840	195.617
180731NF02	pSB1C3_BBa_B0033_mraz	143.368
180731NF03	pSB1C3_BBa_B0033_cbtA	174.778
180731NF04	pSB1C3_BBa_B0032_cbtA	173.534
180731NF05	pSB1C3_BBa_B0033_cspD	110.056
180731NF06	pSB1C3_BBa_B0032_sulA	152.544
180731NF07	pSB1C3_BBa_B0031_sulA	167.888
180731NF08	pSB1C3_oHybB	134.928
180731NF09	pSB1C3_BBa_K206000_BBa_B0034_sulA	139.008

Golden Gate Assembly

A	Calculation	Length [bp]	Density [ng/µL]	Volume [µL]	3MB
	Promotor	161	99.724	0.1113930013	pBAD
	Part 1	1050	62.293	1.163005508	ADH2
	Part 2	1650	91.148	1.24901749	kivD
	Part 3	1590	88.001	1.246640513	LeuA
	Part 4	1100	61.115	1.241871294	LeuB
	Part 5	1410	93.537	1.040081557	LeuC
	Part 6	630	109.97	0.3952738135	LeuD
	Buffer			2
	Mix			1
	pGGA			1
	H₂O_dest			9.552716824

B	Calculation	Length [bp]	Density [ng/µL]	Volume [µL]	Toxin
	Promotor	161	99.724	0.1113930013	pBAD
	Part 1	290	127.138	0.1573817398	BjaIT
	Part 2	210	39.76	0.3644220428	HlyA
	Buffer			2
	Mix			1
	pGGA			1
	H₂O_dest			15.36680322

C	Calculation		Length [bp]		Density [ng/µL]		Volume [µL]		Excretion 1
	Promotor		161		99.724		0.1113930013		pBAD
	Part 1		5164		207.123		1.72024231		HlyB+HlyD+TolC
	Part 2		975		48.8		1.378530924		ompT
	Buffer						2
	Mix						1
	pGGA						1
	H₂O_dest						12.78983376
D		Calculation		Length [bp]		Density [ng/µL]		Volume [µL]		Excretion 2
		Promotor		161		99.724		0.1113930013		pBAD
		Part 1		3872		205.409		1.30061153		HlyB+HlyD
		Part 2		1296		207.256		0.4314491411		TolC
		Part 3		975		48.8		1.378530924		ompT
		Buffer						2
		Mix						1
		pGGA						1
		H₂O_dest						12.7780154

3MB, Toxin, Excretion
37°C	1 min
16°C	1 min
Go to step 1 30x
55°C	5 min

Excretion 1
37°C	1 min
Go to step 1 30x
16°C	1 min

Sequencing

Seq.ID	Lotnumber	Part	Conc. [ng/µL]	Length total [bp]	Part [bp]	Primer	V Part [µL]	V Wasser [µL]
98EC87	180329DW03	pSB1C3-MlcRE	102	341	201	vf2	0.98	4.02
98EC86	180731NF01	pSB1C3-MlcRE-BBa_E0840	195.6	1.225	1.085	vf2	0.51	4.49
98EC90	180731NF01	pSB1C3-MlcRE-BBa_E0840	195.6	1.259	1.085	vr	0.51	4.49
98EC89	180329DW09	pSB1C3-sulA	131.5	650	510	vf2	0.76	4.24
98EC88	180731NF07	pSB1C3-BBa_B0031-sulA	167.9	670	530	vf2	0.6	4.4
98EC91	180329DW21	pSB1C3-BBa_B0034-sulA	75.2	668	528	vf2	1.33	3.67
98EC92	180731NF008	pSB1C3-oHybB	135	313	173	vf2	0.74	4.26
98EC93	180329DW28	pSB1C3-BBa_E0840-oHybB	139.3	1.197	1.057	vf2	0.72	4.28
98EC94	180329DW28	pSB1C3-BBa_E0840-oHybB	139.3	1.231	1.057	vr	0.72	4.28
98EC95	180329DW41	pSB1C3-BBa_K206000-BBa_B0032-sulA	101.3	805	665	vf2	0.99	4.01
98EC96	180731NF09	pSB1C3-BBa_K206000-BBa_B0034-sulA	139	804	664	vf2	0.72	4.28
98EC97	180426NK28	pSB1C3-BBa_B0034-ldhA	85.5	1.194	1054	vf2	1.17	3.83
98EC98	180426NK28	pSB1C3-BBa_B0034-ldhA	85.5	1.228	1054	vr	1.17	3.83
98EC99	180426NK30	pSB1C3-BBa_K206000-BBa_B0031-sulA	80.5	806	666	vf2	1.24	3.76
98ED00	180731NF02	pSB1C3-BBa_B0033-mraz	143.4	615	475	vf2	0.7	4.3
98ED01	180731NF03	pSB1C3-BBa_B0033-cbtA	174.8	532	392	vf2	0.57	4.43
98ED02	180731NF04	pSB1C3-BBa_B0032-cbtA	173.5	534	394	vf2	0.58	4.42
98ED03	180731NF05	pSB1C3-BBa_B0033-cspD	110.1	379	239	vf2	0.91	4.09
98ED04	180531LP05	pSB1C3-BBa_B0032-cspD	53.5	381	241	vf2	1.87	3.13
98ED05	180731NF06	pSB1C3-BBa_B0032-sulA	152.5	669	529	vf2	0.66	4.34
98ED06	180627LP01	pSB1C3-BBa_B0032-ldhA	76	1.195	1055	vf2	1.32	3.68
98ED07	180627LP01	pSB1C3-BBa_B0032-ldhA	76	1.229	1055	vr	1.32	3.68
98ED08	180627LP03	pSB1C3-cbtA	65.5	515	375	vf2	1.53	3.47
98ED09	180703BK01	pSB1C3-BBa_K206000-BBa_B0032-ldhA	153	1.331	1191	vf2	0.65	4.35
98ED10	180703BK01	pSB1C3-BBa_K206000-BBa_B0032-ldhA	153	1.365	1191	vr	0.65	4.35
98ED11	180703BK02	pSB1C3-BBa_K206000-BBa_B0034-ldhA	167	1.330	1190	vf2	0.6	4.4
98ED12	180703BK02	pSB1C3-BBa_K206000-BBa_B0034-ldhA	167	1.364	1190	vr	0.6	4.4
98ED13	180703BK04	pSB1C3-cspD	129	362	222	vf2	0.78	4.22
98ED14	180703BK05	pSB1C3-BBa_B0032-accB	212	630	490	vf2	0.47	4.53
98ED15	180703BK06	pSB1C3-BBa_B0032-mraz	250	617	477	vf2	0.4	4.6
98ED16	180712BK01	pGGA-dapAP-30cbtA-30cspD-30mraz-34sulA	169	1.940	1800	Goldengate_cPCR_r	0.59	4.41
98ED17	180712BK01	pGGA-dapAP-30cbtA-30cspD-30mraz-34sulA	169	-	1800	Goldengate_cPCR_r	0.59	4.41
98ED18	180712BK03	pSB1C3-RnaG120-Operator + MlcRE	93	473	333	vf2	1.08	3.92
98ED19	180724LP01	pSB1C3-BBa_K410000	100	1.657	1517	vf2	1	4
98ED20	180724LP01	pSB1C3-BBa_K410000	100	1.691	1517	vr	1	4

As seen on the table we diluted the template DNA concentration. We added 5 µL Primer (3µM). The total volume were 10 µL.

cPCR of the transformation from 180731

Mastermix for 20 samples:

40 µL 10x Taq buffer
8 µL dNTPs (10 mM)
12 µL MgCl₂
1 µL Taq polymerase
5 µL H₂O_dest
12 µL GGA_cPCR_f
12 µL GGA-cPCR_r

Part	Part	PCR tube
3MB	pBAD
3MB	ADH2	1-4
3MB	kivD	5-8
3MB	leuA	9-12
3MB	leuB	13-16
3MB	leuC	17-20
3MB	leuD	21-24
Toxin	pBAD
Toxin	BjaIT	25-28
Toxin	HlyA
Ex 1	pBAD
Ex 1	TolC	29-32
Ex 1	ompT	33-36
Ex 2	pBAD
Ex 2	HlyB	37-40
Ex 2	TolC	41-44
Ex 2	ompT	44-48

PCR program:

95°C	2 min
95°C	30 s
55°C	30 s
72°C	3 min
Go to step 2 30x
72°C	5 min

PCR for GGAs and Basic Parts

Part	tube	fw_Primer	rv_Primer	template	pred. Length	concentration [ng/µL]
Bsa_accD	1	Bsa_accD_f	Bsa_accD_rv	180730BK04	950	11.558
Bsa_kivD	2	Bsa_kivD_f	Bsa_kivD_r	180730BK07	1700	11.619
Bsa_pBAD	3	Bsa_dapAP_f	Bsa_pBAD_r	180730BK14	200	16.782
Bsa_HlyB	4	Bsa_HlyB_f	Bsa_HlyB_r	180730BK15	3850	7.68
Bsa_OmpT	5	Bsa_OmpT_f	Bsa_OmpT_r	180730BK16	1000	9.895
Bsa_34-cbtA	6	Bsa_34-cbtA_f	cbtA_Bsa_r	180622NB07	400	58.154
Bsa_34-cspD	7	Bsa_34-cspD_f	cspD_Bsa_r	180622NB04	250	65.224
Bsa_34-mraz	8	Bsa_34-mraz_f	mraz_Bsa_-r	180622NB08	500	66.294
Bsa_34-sulA	9	Bsa_34-sulA_f	sulA_Bsa_r	180329DW09	580	87.334
OmpT	10	OmpT_fz	OmpT_r2	e.coli	1000	60.291
33_killR	11	33_killR_f	killR_r	e.coli	280	58.983

Mastermix:

20 µL H₂O_dest
25 µL Q5 Master Mix
5 µL of each Primer

PCR Program:

		Tube 4
98°C	2 min	20 s
98°C	10 s
55°C	20s
72°C	1:30 min	2:30 min
Go to step 2 30x
72°C	2 min

cPCR of trafo on 180807

We made two Master mixes.

Master Mix 1 for 25 samples:

50 µL Dream Taq Buffer
10 µL dNTPs
15 µL MgCl₂
25 µL Dream Tag Polymerase
15 µL GGA_cPCR_fw
15 µL GGA_cPCR_rv
75 µL H₂O_dest

The plates was sealed with Parafilm and stored in the fridge.

Master Mix 2:

40 µL Dream Taq Buffer
8 µL dNTPs
12 µL MgCl₂
1 µL Dream Taq Polymerase
12 µL GGA_cPCR_fw
12 µL psal_pBAD_rv
315 µL H₂O_dest

PCR-Program:

PCR 1		PCR 2
95°C	3 min	95°C	3 min
95°C	30 s	95°C	30 s
55°C	30 s	55°C	30 s
72°C	8 min	72°C	1 min
Go to step 2 30x		Go to step 2 30x
72°C	10 min	72°C	2 min

New Labels

PCR 1:

BK01 = 3MB 1-5
BK02 = EX 1-5
BK03 = MA 1-5
BK04 = GI 1-5 à No PCR 2-sample, because the plasmid is too small.

PCR 2:

BK01 = 3MB 1-5
BK02 = EX 1-5
BK03 = MA 1-5

cPCR of 3MB#1, Myristic Acid#1 (MA#1), and Myristic Acid#4 (MA#4)

Master Mix for 20 samples:

40 µL Dream Taq Buffer
8 µL dNTPs
12 µL MgCl₂
1 µL Dream Taq
2 µL of each primer
315 µL H₂O_dest

Tube	Part	Part-components	Primer
1	3MB	ADH2	fw-GGa-cPCR
2	3MB	kivD, AD2	fw-GGa-cPCR
3	3MB	LeuA, kivD, ADH2	fw-GGa-cPCR
4	3MB	LeuB, LeuC, LeuD	GGA-cPCR-rv
5	3MB	LeuC, LeuD	GGA-cPCR-rv
6	3MB	LeuD	GGA-cPCR-rv
7	MA#1	accA	GGA-cPCR-fw
8	MA#1	accB, accA	GGA-cPCR-fw
9	MA#1	accC, accD, tesA	GGA-cPCR-rv
10	MA#1	accD, tesA	GGA-cPCR-rv
11	MA#1	tesA	GGA-cPCR-rv
12	MA#4	accA	GGA-cPCR-fw
13	MA#4	accB, accA	GGA-cPCR-fw
14	MA#4	accC, accD, tesA	GGA-cPCR-rv
15	MA#4	accD, tesA	GGA-cPCR-rv
16	MA#4	tesA	GGA-cPCR-rv

cPCR Program:

95°C	3 min
95°C	30 s
55°C	30 s
72°C	5 min
Go to step 2 30x
72°C	10 min

PCR for 3A-Assembly + GGA

Part	Tube	fw-Primer	rv-Primer	Template	Length [bp]
pSB1C3	1	SB-prep-2Ea	SB-prep-3P1	pSB1C3-I20270	2000
accD	2	Pre_Syn_f	Syn_Suf_r	IDT	950
tesA	3	Pre_tesA_f	tesA_Suf_r	e.coli	650
Bsa_accD	4	-	-	180806DK01	950
Bsa_kivD	5	-	-	180806DK02	1650
Bsa_HlyB	6	-	-	180806DK04	3800
Bsa_OmpT	7	-	-	180806DK05	1000

Mastermix:

25 µL Q5 Mastermix
20 µL H₂O_dest
5 µL of each Primer
25 µL template

Note: We made a new Mastermix, because it contained white flake. Mastermix was used by sample 4-7.

PCR-Program:

98°C	2 min	20 s (HyB + tesA)
98°C	10 s
55°C	20 s
72°C	1:20 min	2 min (Hyb + tesA)
Go to step 2 30x
72°C	2 min

Restriction of remaining 3A-Assembly + GGA

Calculation	Länge (bp)	Dichte (ng/µL)	Volumen (µL)	3MB
Promotor	161	99	0.5	pBAD
Part 1	1050	62.293	2.90751377	ADH2
Part 2	1650	91	3.127622148	kivD
Part 3	1590	88.001	3.116601282	LeuA
Part 4	1100	61.115	3.104678235	LeuB
Part 5	1410	93.537	2.600203891	LeuC
Part 6	630	109.97	0.9881845337	LeuD
Buffer			2
Mix			1
pGGA			1
H2O			0



Calculation	Länge (bp)	Dichte (ng/µL)	Volumen (µL)	Excretion
Promotor	161	99	0.5610381645	pBAD
Part 1	3872	205	6.516032042	HlyB+HlyD
Part 2	1296	207.256	2.157245705	TolC
Part 3	975	49	6.864521338	ompT
Buffer			2
Mix			1
pGGA			1
H2O			0

Calculation	Länge (bp)	Dichte (ng/µL)	Volumen (µL)	Myristic Acid
Promotor	161	99.724	0.569650063	pBAD
Part 1	993	174.035	1.968404615	accA
Part 2	489	92.21	1.829500619	accB
Part 3	1358	108.9	4.302031775	accC
Part 4	925	189	1.688424527	accD
Part 5	679	111.71	2.096908336	tesA
Buffer			2
Mix			1
pGGA			1
H2O			3.557765122

Calculation	Länge (bp)	Dichte (ng/µL)	Volumen (µL)	Growth Inhibition
Promotor	161	99	0.5610381645	pBAD
Part 1	410	58.154	2.432237546	34cbtA
Part 2	260	65.224	1.375205632	34cspD
Part 3	500	66.294	2.601941356	34mraZ
Part 4	565	87.334	2.231859337	34sulA
Buffer			2
Mix			1
pGGA			1
H2O			6.797717965

Tube

1

3

5

7

8

9

10

11

12

13

14

Tube

6

8

10

Part

pSB1C3-BBa_K206000

pSB1C3-I13453

pSB1C3-NOTMlcRE

pSB1C3-E0840

pSB1C3-BBa_B0032-cbtA

pSB1C3-BBa_B0033-cbtA

pSB1C3-BBa_B0032-cspD

pSB1C3-BBa_B0033-cspD

pSB1C3-BBa_B0032-mraZ

pSB1C3-BBa_B0033-mraZ

Part

pSB1C3-BBa_K206001

pSB1C3-I13454

pSB1C3-NOTMlcRE

Lot#

180307NK01

180802BK02

180712BK03

180627LP02

180731NF04

180731NF03

180531LP05

180731NF05

180703BK06

180731NF02

Lot#

180307NK02

180802BK03

180712BK04

ρ

47 ng/µL

90 ng/µL

93 ng/µL

92 ng/µL

174 ng/µL

175 ng/µL

54 ng/µL

110 ng/µL

250 ng/µL

143 ng/µL

ρ

48 ng/µL

91 ng/µL

94 ng/µL

Use

vector

Use

vector

target m

2000 ng

target m

Volumes

DNA

25.00 µL

8.00 µL

12.50 µL

12.81 µL

11.00 µL

10.00 µL

11.00 µL

10.00 µL

Volumes

DNA

26.00 µL

9.00 µL

13.50 µL

NEBuffer 2.1

3.00 µL

2.00 µL

NEBuffer 2.2

EcoRI-HF

0.50 µL

EcoRI-HF

XbaI

0.50 µL

XbaI

SpeI

0.50 µL

SpeI

0.50 µL

PstI

0.50 µL

PstI

0.50 µL

FastAP

0.50 µL

FastAP

H2O

0.50 µL

8.50 µL

4.00 µL

3.69 µL

6.00 µL

7.00 µL

6.00 µL

7.00 µL

H2O

ad

30.00 µL

20.00 µL

ad

Länge Produkt

ρ final

39 ng/µL

36 ng/µL

58 ng/µL

59 ng/µL

51 ng/µL

87 ng/µL

29 ng/µL

55 ng/µL

125 ng/µL

72 ng/µL

ρ final

Gelextraction of the restrictions and PCR-Product accD

The Gelextraction was made by GeneJET Gelextraction Kit. After that we measuered the DNA-concentration via Nano Drop.

Tube	Part	DNA-concentration [ng/µL]
1	pSB1C3-K206000	5.3
3	pSB1C3-I13453	10.6
5	pSB1C3-NOTMlcRE	8.3
7	pSB1C3-E0840	11.3
8	E0840	2
9	32-cbtA	1.8
10	33-cbtA	4.1
11	32-cspD	10
12	33-cspD	3.3
13	32-mraZ	9.6
14	33-mraZ	32.5
23	AOX1	20.7
	accD (180814OM01)	7.6

GGA Assembly + Ligation

GGA Assembly program:

37°C	5 min
16°C	10 min
Go to step 1 40x
55°C	5 min

Tube	Part
1	3MB
2	Excretion
3	Myristic Acid
4	Growth Inhibition

The ligation ran for 16 h by 16°C.

Tube

1

L2

L3

L4

L5

L6

L7

L8

L9

L10

L11

L12

L13

L14

L15

L16

L17

L18

L19

L20

L21

Tube

pSB1C3-BBa_K206000-pSB1C3-BBa_B0032-cbtA

pSB1C3-BBa_K206000-pSB1C3-BBa_B0033-cbtA

pSB1C3-BBa_K206000-pSB1C3-BBa_B0032-cspD

pSB1C3-BBa_K206000-pSB1C3-BBa_B0033-cspD

pSB1C3-BBa_K206000-pSB1C3-BBa_B0032-mraZ

pSB1C3-BBa_K206000-pSB1C3-BBa_B0033-mraZ

pSB1C3-I13453-pSB1C3-BBa_B0032-cbtA

pSB1C3-I13453-pSB1C3-BBa_B0033-cbtA

pSB1C3-I13453-pSB1C3-BBa_B0032-cspD

pSB1C3-I13453-pSB1C3-BBa_B0033-cspD

pSB1C3-I13453-pSB1C3-BBa_B0032-mraZ

pSB1C3-I13453-pSB1C3-BBa_B0033-mraZ

pSB1C3-NOTMlcRE-pSB1C3-E0840

pSB1C3-E0840-

pSB1C3-

pSB1C3-BBa_K206000-

pSB1C3-I13453-

pSB1C3-E0840-

pSB1C3-

Tube

pSB1C3-BBa_K206000

pSB1C3-I13453

pSB1C3-NOTMlcRE

pSB1C3-E0840

pSB1C3

pSB1C3-BBa_K206000

pSB1C3-I13453

pSB1C3-E0840

pSB1C3

Tube

1

3

5

7

17

1

3

7

17

Tube

5 ng/µL

11 ng/µL

8 ng/µL

59 ng/µL

10 ng/µL

5 ng/µL

11 ng/µL

10 ng/µL

Tube

2200 bp

2500 bp

2000 bp

2200 bp

2800 bp

2000 bp

Tube

2.50 µL

3.00 µL

2.00 µL

2.50 µL

3.00 µL

2.00 µL

2.87 µL

1.67 µL

1.50 µL

1.57 µL

3.64 µL

4.44 µL

Tube

pSB1C3-BBa_B0032-cbtA

pSB1C3-BBa_B0033-cbtA

pSB1C3-BBa_B0032-cspD

pSB1C3-BBa_B0033-cspD

pSB1C3-BBa_B0032-mraZ

pSB1C3-BBa_B0033-mraZ

pSB1C3-BBa_B0032-cbtA

pSB1C3-BBa_B0033-cbtA

pSB1C3-BBa_B0032-cspD

pSB1C3-BBa_B0033-cspD

pSB1C3-BBa_B0032-mraZ

pSB1C3-BBa_B0033-mraZ

pSB1C3-E0840

Tube

9

10

11

12

13

14

9

10

11

12

13

14

8

21

22

24

23

25

26

Tube

2 ng/µL

4 ng/µL

10 ng/µL

3 ng/µL

10 ng/µL

33 ng/µL

2 ng/µL

4 ng/µL

10 ng/µL

3 ng/µL

10 ng/µL

33 ng/µL

2 ng/µL

21 ng/µL

Tube

400 bp

250 bp

500 bp

400 bp

250 bp

500 bp

800 bp

1000 bp

1100 bp

400 bp

250 bp

Tube

9.00 µL

6.00 µL

10.00 µL

7.23 µL

6.00 µL

9.00 µL

5.32 µL

9.00 µL

7.23 µL

9.00 µL

Tube

2.00 µL

1.00 µL

2.00 µL

1.00 µL

2.00 µL

1.00 µL

2.00 µL

1.00 µL

Tube

0.50 µL

Tube

6.00 µL

8.50 µL

4.50 µL

8.27 µL

0.50 µL

6.00 µL

0.18 µL

5.50 µL

8.27 µL

6.50 µL

5.63 µL

8.50 µL

6.83 µL

7.00 µL

6.93 µL

4.86 µL

4.06 µL

Tube

20.00 µL

10.00 µL

20.00 µL

10.00 µL

20.00 µL

10.00 µL

20.00 µL

10.00 µL

Solving the Nootkatone DNA of Düsseldorf

We cut circles out of paper with the seperated plasmids. Then we put them in 1.5 µL tubes and overlayed with 100 µL Elution Buffer. After that we vortexed the tubes, incubated for 5 min and vortexed again. Afterwards we did a transformation and stored the residue of the mix (Plasmid + Buffer) at -20°C (180814BK01-04).

	#	Sample ID	User name	Date and time	DNA-concentration [ng/µL]	A260 (Abs)	A280 (Abs)	260/280	260/230	Sample Type	Factor	Abx
	208	h2o	Nanodrop	02.08.2018 11:57:03	0.7	0.015	0.02	0.74	0.16	DNA	50
1	209	ADH	Nanodrop	02.08.2018 11:57:41	392.3	7.846	4.178	1.88	1.84	DNA	50	Cam
2	210	ValS	Nanodrop	02.08.2018 11:58:22	268.6	5.373	2.935	1.83	1.37	DNA	50	Cam
3	211	AIPLF	Nanodrop	02.08.2018 11:58:52	678	13.559	7.325	1.85	2.12	DNA	50	Amp
4	212	AIPLF-pts1	Nanodrop	02.08.2018 11:59:21	486.1	9.721	5.25	1.85	1.89	DNA	50	Amp

Miniprep of the overnight cultures from 180816

Lotnumber	Part	DNA-concentration [ng/µL]
180817LD01	cPCR 1	117
180817LD02	cPCR 2	132
180817LD03	cPCR 10	66
180817LD04	cPCR 16	110
180817LD05	cPCR 18	181
180817LD06	cPCR 24	113
180817LD07	cPCR 27	266
180817LD08	cPCR 35	10
180817LD09	cPCR 38	191
180817LD10	pSB1C3-E0240	163
180817LD11	pSB1C3-ADH #1	185
180817LD12	pSB1C3-ValS #2	79
180817LD13	pSB1C3-AlPLF #3	453
180817LD14	pSB1C3-AlPLF-pts1 #4	326

Restriction

	Tube	1	2	3	4	5	6	7	8	9
	Part	pSB1C3-I13453	pSB1C3-NOTMlcRE	pSB1C3-E0240	pSB1C3-BBa_B0032-cspD	pSB1C3-BBa_B0032-mraZ	pSB1C3-AOX1	pSB1C3	OmpT	BBa_J45503
	Lot#	180822BK01	180822BK02	180817LD10	180531LP05	180703BK06	180726LP02	180814LP01	180806BK10	180625NiF12
	ρ	115 ng/µL	119 ng/µL	163 ng/µL	54 ng/µL	250 ng/µL	114 ng/µL	101 ng/µL	60 ng/µL	45 ng/µL
	Use
	target m

Volumes	DNA	0.87 µL	0.84 µL	0.61 µL	1.87 µL	0.40 µL	0.88 µL	0.99 µL	1.66 µL	2.24 µL
	NEBuffer 2.1	1.00 µL	1.00 µL	1.00 µL	1.00 µL	1.00 µL	1.00 µL	1.00 µL	1.00 µL	1.00 µL
	EcoRI-HF							0.50 µL	0.50 µL	0.50 µL
	XbaI			0.50 µL	0.50 µL	0.50 µL	0.50 µL
	SpeI	0.50 µL	0.50 µL
	PstI	0.50 µL	0.50 µL	0.50 µL	0.50 µL	0.50 µL	0.50 µL	0.50 µL	0.50 µL	0.50 µL
	FastAP	0.50 µL	0.50 µL	0.50 µL	0.50 µL	0.50 µL	0.50 µL
	H2O	6.63 µL	6.66 µL	6.89 µL	5.63 µL	7.10 µL	6.62 µL	7.01 µL	6.34 µL	5.76 µL
	ad	10.00 µL	10.00 µL	10.00 µL	10.00 µL	10.00 µL	10.00 µL	10.00 µL	10.00 µL	10.00 µL

	Länge Produkt

	ρ final	10 ng/µL	10 ng/µL	10 ng/µL	10 ng/µL	10 ng/µL	10 ng/µL	10 ng/µL	10 ng/µL	10 ng/µL

Ligation

Tube		L1	L2	L3	L4	L5	L6
Product		pSB1C3-I13453-pSB1C3-BBa_B0032-cspD	pSB1C3-I13453-pSB1C3-BBa_B0032-mraZ	pSB1C3-I13453-pSB1C3-AOX1	pSB1C3-NOTMlcRE-pSB1C3-E0240	pSB1C3-OmpT	pSB1C3-BBa_J45503

Vector	Vector	pSB1C3-I13453	pSB1C3-I13453	pSB1C3-I13453	pSB1C3-NOTMlcRE	pSB1C3	pSB1C3
	Tube	1	1	1	2	7	7
	ρ	10 ng/µL	10 ng/µL	10 ng/µL	10 ng/µL	10 ng/µL	10 ng/µL
	Länge	2200 bp	2200 bp	2200 bp	2500 bp	2070 bp	2070 bp
	V	4.40 µL	3.01 µL	1.55 µL	2.21 µL	1.71 µL	4.08 µL
Part 1	Part 1	pSB1C3-BBa_B0032-cspD	pSB1C3-BBa_B0032-mraZ	pSB1C3-AOX1	pSB1C3-E0240	OmpT	BBa_J45503
	Tube	4	5	6	3	8	9
	ρ	10 ng/µL	10 ng/µL	10 ng/µL	10 ng/µL	10 ng/µL	10 ng/µL
	Länge	280 bp	510 bp	1200 bp	880 bp	1000 bp	300 bp
	V	5.60 µL	6.99 µL	8.45 µL	7.79 µL	8.29 µL	5.92 µL
Part 2	Part 2
	Tube
	ρ
	Länge
	V
T4 Buffer		2.00 µL	2.00 µL	2.00 µL	2.00 µL	2.00 µL	2.00 µL
T4 Ligase		0.50 µL	0.50 µL	0.50 µL	0.50 µL	0.50 µL	0.50 µL
H2O		7.50 µL	7.50 µL	7.50 µL	7.50 µL	7.50 µL	7.50 µL
ad		20.00 µL	20.00 µL	20.00 µL	20.00 µL	20.00 µL	20.00 µL

Characterization of sulA (OD-measure)

We measured the growth curve at 600 nm.

Measure of the overnight cultures:

Cultures	OD
DH5a	0.6
31-sulA	1.05
32-sulA	0.92
34-sulA	0.97

We had to adjust the OD of each culture with the following formula.

Requested start-OD: 0.01

0.01/0.06 * 35 mL = 0.583 mL of the overnight cultures in 35 mL LB-medium.

After preparing the cultures they were stored at 37°C, 170 rpm and measured every half hour.

Time [h]	DH5a	31-sulA	32-sulA	34-sulA
0:30	0.014	0.014	0.018	0.022
1:00	0.05	0.043	0.035	0.032
1:30	0.136	0.084	0.094	0.089
2:00	0.253	0.178	0.203	0.194
2:30	0.554	0.476	0.519	0.497
3:00	0.878	0.809	0.855	0.843
3:30	1.129	1.145	1.159	1.112

After 2 h we added Arabinose to the samples.

Calculation:

V = 30 mL

c = 60 µM

M = 150.13 g/mol

m = 1.8 x10^-6 mol

m = c * V * M = 0.225 mg Arabinose to each culture.

Measure was canceled, because we had not important results. We had a permanent growth and had not added the Arabinose at the beginning.

Repetition of sulA characterization

Culture	OD 600	Timepoint	Requested start-OD: 0.01
DH5a	3.28	1:32 pm	0.108
BBa-B0031-sulA	3.456	1:34 pm	0.126
BBa-B0034-sulA	4.192	1:42 pm	0.11

We had to adjust the OD of each culture. We added 100 mL cultures in LB-medium (+ Camp in sulA) into 300 mL-flasks and shacked them by 170 rpm and 37°C. After 2 hours we added 5 ml 100%-L-Arabinose.

	Time [min]	DH5a	BBa-B0031-sulA	BBa-B0034-sulA
t₀	0	0.108	0.126	0.11
t₁	60	0.222	0.324	0.284
t₂	90	0.408	0.592	0.532
t₃	120	0.82	1.156	1.096
t₄	180	1.852	3.138	3.262

After the measurement we microscoped the cultures and took some pictures. We had not noticed differences between DH5a and sulA.

PCR of multiple Parts

PCR were prepapared as followed:

Tube	Part	Primer-fw	Primer-rv
1	pre-31-sulA-suf	31-sulA-Fw	SulA-Suf-Rev
2	pre-oHybB-suf	Pre-HybB-Fw	HybB-Suf-Rev
3	pre-34-sulA-suf	34-sulA-Fw	SulA-Suf-Rev
4	pre-32-sulA-suf	32-sulA-Fw	SulA-Suf-Rev
5	pre-32-cspD-suf	Pre_cspD_f	cspD_Suf_r
6	pre-32-LdhA-suf	32-Idha-Fw	ldhA-Suf-Rev
7	pre-cspD-suf	Pre-cspD-f	cspD-Suf-Rev

Mastermix for 10 approaches were prepared as shown:

50 µL	Q5 Mastermix
5 µL	Template DNA (DH5α)
35 µL	H₂O

This was aliquoted into 0.5 mL reaction tube to total volume of 9 µL. To each aliquot were added the specific primer pair, 0.5 µL forward and 0.5 µL reverse primer.

The PCR program was done as shown in the next table.

1	98 °C	30 s
2	98 °C	10 s
3	63 °C	30 s
4	72 °C	30 s
5	go to step 2 30 times
6	72 °C	2 min

For the analytic of the PCR products we used a 1 % agarose gel. 2 µL of the PCR product were loaded on the gel.

Because we used undiluted primers in this approach, we repeated the PCR with a new mastermix, the right primers and the same PCR program. New mastermix is shown in the following table.

250 µL	Q5 Mastermix
25 µL	Template DNA (DH5α)
175 µL	H₂O

This was aliquoted into 0.5 mL reaction tube to total volume of 4.5 µL. To each aliquot were added the specific primer pair, 2.5 µL forward and 2.5 µL reverse primer. Here we used a wrong ratio of primer to Q5 mastermix. So, we repeated this experiment.

https://static.igem.org/mediawiki/2018/7/75/T--Hamburg--180829_PCR_%28nicht_erfolgreich%29_%282%29.JPG

PCR repetition from 180829

Mastermix for 50 samples:

250 µL
25 µL of each primer
25 µL Template
175 µL H₂O_dest

Tube	Part	Length [bp]
1	Pre-31-sulA-suf	530
2	Pre-32-sulA-suf	529
3	Pre-34-sulA-suf	528
4	Pre-oHybB-suf	173
5	Pre-32-ldhA-suf	1055
6	Pre-cspD-suf	222
7	Pre-32-cspD-suf	241

PCR program:

98°C	2 min
98°C	40 s
55°C	20 s
72°C	1:20 min
Go to step 2 30x
72°C	2 min

Restriction of multiple parts

Part Lot-No.

               pSB1C3-RNAG120-MlcrE 180822BK02
               pSB1C3-oHybB                                 180731NF08
               pSB1C3-E0240                                  180817LD10
               pSB1C3-E0840                                  180627LP02
Reaction mix:

	pSB1C3-RNAG120-MlcRE	pSB1C3-oHybB	pSB1c3-E0240	pSB1C3-E0840
DNA	1.69 µL	1.48 µL	1.23 µL	2.17 µL
NE-Buffer 2.1	1 µL	1 µL	1 µL	1 µL
SpeI	0.5 µL	0.5 µL	-	-
PstI	0.5 µL	0.5 µL	0.5 µL	0.5 µL
XbaI	-	-	0.5 µL	0.5 µL
FastAP	0.5 µL	0.5 µL	0.5 µL	0.5 µL
H2O	5.81 µL	6.02 µL	6.27 µL	5.33 µL

Restriction ran for 1 h at 37 °C, followed by 20 min at 80 °C.

Separation of gfp-parts by gel electrophoresis with a 1 % agarose gel, followed by a gel extraction.

Resulting concentrations were:

Part c [ng/µL]

               pSB1C3-RNAG120-MlcrE 75.43
               pSB1C3-oHybB                                 259.27
               pSB1C3-E0240                                  1
               pSB1C3-E0840                                  2

The gfp parts showed very low concentrations and were discarded.

Repeating the PCR from 30.08.18

PCR was performed as follows.

5xPhusion HF Buffer	70 µL
dNTPs	7 µL
DNA (E. coli)	7µL
Phusion Polymerase	3.5 µL
Fw primer	1 µL
Rv prmer	1 µL
ad 350 µL H₂O	248.5 µL

The PCR programme was carried out as follows.

Step	Temperature	Time [s]
1	98 °C	180
2	98 °C	10
3	55 °C	30
4	72 °C	120
go to step 2 (30x)
5	72 °C	300

The following primer pairs were used.

1	31sulA	(31-sulA-Fw, 31-sulA-Suf-Rv)
2	32sulA	(32-sulA-Fw, 32-sulA-Suf-Rv)
3	34sulA	(34-sulA-Fw, 34-sulA-Suf-Rv)
4	HybB	(pre-HybB-fw, pre-HybB-Suf-Rv)
5	32IdhA	(32IdhA-fw, 32IdhA-Suf-Rv)
6	cspD	(pre-cspD-fw, pre-csp-Suf-Rv)

The PCR results were analysed in an agarose gel (1%, 80 V, 45 min).

1	2	3	4	5	6
31sulA	32sulA	34sulA	HybB	32IdhA	cspD
positive	positive	positive	positive	negative	positive

Restriction of the PCR from 30.08.18

The PCR products were restricted according to the following table. Afterwards, the DNA concentration was measured via nano drop.

	31sulA	32sulA	34sulA	HybB	cspD
NEB2.1 [µL]	2	2	2	2	2
DNA [µL]	4	4	4	4	4
EcoRI [µL]				0.5	0.5
XbaI [µL]	0.5	0.5	0.5
SpeI [µL]				0.5
pstI [µL]	0.5	0.5	0.5		0.5
H₂O [µL]	13	13	13	13	13
concentration [ng/µL]	129.38	183.26	169.36	210.06	166.14

Restriction

Tube

1

2

3

4

5

6

7

9

10

11

12

13

14

15

Part

MlcRE

sulA

31sulA

psB1C3-E0840

oHybB

pSB1C3-BBa_K206000

pSB1C3

32sulA

34sulA

cspD

pSB1C3-BBa_B0032

cspD

psB1C3-E0840

pSB1C3-NOT-MlcRE

Lot#

180827LP03

180827LP02

180905AW01

180315NB01

aus PCR

180829LD01

180814LP01

180905AW02

180905AW03

aus PCR

180514NB07

aus PCR

180315NB01

180822BK02

ρ

63 ng/µL

71 ng/µL

28 ng/µL

128 ng/µL

69 ng/µL

115 ng/µL

101 ng/µL

40 ng/µL

43 ng/µL

48 ng/µL

81 ng/µL

48 ng/µL

128 ng/µL

118 ng/µL

Use

single insert

assembly insert 2

vector

single insert

vector

assembly insert 2

single insert

vector

assembly insert 2

vector

target m

100 ng

2000 ng

100 ng

2000 ng

100 ng

2000 ng

100 ng

2000 ng

Volumes

DNA

1.60 µL

1.40 µL

3.57 µL

7.00 µL

1.45 µL

5.00 µL

2.50 µL

2.33 µL

2.08 µL

5.00 µL

2.08 µL

0.78 µL

7.00 µL

NEBuffer 2.1

1.00 µL

EcoRI-HF

0.20 µL

0.40 µL

0.20 µL

XbaI

0.20 µL

0.40 µL

0.20 µL

SpeI

0.20 µL

PstI

0.20 µL

FastAP

0.50 µL

H2O

7.00 µL

7.20 µL

5.03 µL

0.70 µL

7.15 µL

3.10 µL

6.10 µL

6.27 µL

6.52 µL

3.10 µL

6.52 µL

7.82 µL

1.10 µL

ad

10.00 µL

Länge Produkt

252 bp

556 bp

565 bp

2948 bp

224 bp

2200 bp

2070 bp

565 bp

245 bp

2083 bp

245 bp

904 bp

2697 bp

ρ final

10 ng/µL

90 ng/µL

10 ng/µL

58 ng/µL

51 ng/µL

10 ng/µL

40 ng/µL

10 ng/µL

83 ng/µL

Ligation

Tube		L1	L2	L5	L6	L7	L9
Product		pSB1C3-MlcRE	pSB1C3-sulA	pSB1C3-BBa_K206000-31sulA	pSB1C3-BBa_K206000-32sulA	pSB1C3-BBa_K206000-34sulA	pSB1C3-BBa_B0032-cspD

Vector	Vector	pSB1C3	pSB1C3	pSB1C3-BBa_K206000	pSB1C3-BBa_K206000	pSB1C3-BBa_K206000	pSB1C3-BBa_B0032
	Tube	7	7	6	6	6	12
	ρ	51 ng/µL	51 ng/µL	58 ng/µL	58 ng/µL	58 ng/µL	40 ng/µL
	Länge	2070 bp	2070 bp	2200 bp	2200 bp	2200 bp	2083 bp
	V	0.89 µL	0.54 µL	0.49 µL	0.49 µL	0.49 µL	1.14 µL
Part 1	Part 1	MlcRE	sulA	31sulA	32sulA	34sulA	cspD
	Tube	1	2	3	9	10	13
	ρ	10 ng/µL	10 ng/µL	10 ng/µL	10 ng/µL	10 ng/µL	10 ng/µL
	Länge	252 bp	556 bp	565 bp	565 bp	565 bp	245 bp
	V	5.49 µL	7.29 µL	7.20 µL	7.20 µL	7.20 µL	5.40 µL
Part 2	Part 2
	Tube
	ρ
	Länge
	V
T4 Buffer		1.00 µL	1.00 µL	1.00 µL	1.00 µL	1.00 µL	1.00 µL
T4 Ligase		0.50 µL	0.50 µL	0.50 µL	0.50 µL	0.50 µL	0.50 µL
H2O		2.12 µL	0.68 µL	0.82 µL	0.82 µL	0.82 µL	1.95 µL
ad		10.00 µL	10.00 µL	10.00 µL	10.00 µL	10.00 µL	10.00 µL


Länge gesamter Part		252 bp	556 bp	695 bp	695 bp	695 bp	258 bp
		597 bp	901 bp	1040 bp	1040 bp	1040 bp	603 bp

Restriction

	Tube	1	2	3	4	5	6	7	8
	Part	pSB1C3-BBa_K206000	pSB1C3	pSB1C3-BBa_I13504	34-AOX1	pSB1C3-BBa_E0840	pSB1C3-BBa_E0840	pSB1C3_RNAG120_MlcRE	pSB1C3_oHybB
	Lot#	180829LD01	180906LP02	180208BK04	180906LP01	180627LP02	180627LP02	180822BK02	180731NF08
	ρ	115 ng/µL	138 ng/µL	141 ng/µL	84 ng/µL	91 ng/µL	91 ng/µL	119 ng/µL	135 ng/µL
	Use	vector	vector	vector	single insert	vector	single insert
	target m	2000 ng	2000 ng	2000 ng	100 ng	2000 ng	100 ng	200 ng	200 ng

Volumes	DNA	16.60 µL	14.49 µL	5.00 µL	1.19 µL	3.00 µL	1.10 µL	1.69 µL	1.48 µL
	NEBuffer 2.1	2.00 µL	2.00 µL	1.00 µL	1.00 µL	1.00 µL	1.00 µL	1.00 µL	1.00 µL
	EcoRI-HF					0.20 µL	0.20 µL
	XbaI		0.40 µL		0.20 µL	0.20 µL
	SpeI	0.40 µL		0.20 µL				0.50 µL	0.50 µL
	PstI	0.40 µL	0.40 µL	0.20 µL	0.20 µL		0.20 µL	0.50 µL	0.50 µL
	FastAP	0.50 µL	0.50 µL	0.50 µL		0.50 µL		0.50 µL	0.50 µL
	H2O	0.10 µL	2.21 µL	3.10 µL	7.41 µL	5.10 µL	7.50 µL	5.81 µL	6.02 µL
	ad	20.00 µL	20.00 µL	10.00 µL	10.00 µL	10.00 µL	10.00 µL	10.00 µL	10.00 µL

	Länge Produkt	2200 bp	2070 bp	2200 bp	1200 bp	2948 bp	878 bp	2697 bp	2326 bp

	ρ final	96 ng/µL	100 ng/µL	71 ng/µL	10 ng/µL	27 ng/µL	10 ng/µL	20 ng/µL	20 ng/µL

								"Tube 1"	"Tube 2"
								vom 4.9.

Ligation

Tube		L1	L2	L3	L4	L5
Product		pSB1C3-BBa_K206000-34-AOX1	pSB1C3-34-AOX1	pSB1C3-BBa_I13504-34-AOX1	pSB1C3_RNAG120_MlcRE-pSB1C3-BBa_E0840	pSB1C3_oHybB-pSB1C3-BBa_E0840

Vector	Vector	pSB1C3-BBa_K206000	pSB1C3	pSB1C3-BBa_I13504	pSB1C3_RNAG120_MlcRE	pSB1C3_oHybB
	Tube	1	2	3	7	8
	ρ	96 ng/µL	100 ng/µL	71 ng/µL	20 ng/µL	20 ng/µL
	Länge	2200 bp	2070 bp	2200 bp	2697 bp	2326 bp
	V	0.16 µL	0.15 µL	0.22 µL	0.50 µL	0.50 µL
Part 1	Part 1	34-AOX1	34-AOX1	34-AOX1	pSB1C3-BBa_E0840	pSB1C3-BBa_E0840
	Tube	4	4	4	6	6
	ρ	10 ng/µL	10 ng/µL	10 ng/µL	10 ng/µL	10 ng/µL
	Länge	1200 bp	1200 bp	1200 bp	878 bp	878 bp
	V	8.45 µL	8.53 µL	8.45 µL	5.00 µL	5.00 µL
Part 2	Part 2
	Tube
	ρ
	Länge
	V
T4 Buffer		2.00 µL	2.00 µL	2.00 µL	1.00 µL	1.00 µL
T4 Ligase		0.50 µL	0.50 µL	0.50 µL	0.50 µL	0.50 µL
H2O		8.89 µL	8.82 µL	8.83 µL	3.00 µL	3.00 µL
ad		20.00 µL	20.00 µL	20.00 µL	10.00 µL	10.00 µL


Länge gesamter Part		1330 bp	1200 bp	1330 bp	1505 bp	1134 bp
plus VR VF2		1675 bp	1545 bp	1675 bp	1850 bp	1479 bp

cPCR oft the ligation form 06.09.18

On all plates except from 180906LP05 and 180906LP04 colonies are grown. Plates 180906LP05 and 180906LP04 were further incubated at 37 °C.

PCR preparation	1x	47x
10x Taq Buffer	2 µL	94 µL
dNTPs 10 mM	0.4 µL	18 µL
50 mM MgCl₂	0.6 µL	28.2 µL
fw primer	0.6 µL	28.2 µL
rv primer	0.6 µL	28.2 µL
Taq polymerase	0.1 µL	4.7 µL
H₂O ad 20 µL	14.5 µL	681.5 µL

The PCR programme was carried out as follows.

Step	Temperature	Time [s]
1	95 °C	600
2	95 °C	30
3	55 °C	30
4	72 °C	120
go to step 2 (30x)
5	72 °C	300

Minipreparation of ONC from 06.09.18

A minipreparation of 180907NiF03 and 180907NiF04 was performed using GeneJET Plasmid Miniprep Kit #K0503 by Thermo Scientific following protocol A. Afterwards, the concentration of DNA was measured via nano drop.

Minipreparation	Concentration (ng/µL)
180907NiF03	83.791
180907NiF04	85.965

180907-2.png

Control of the gel electrophoresis is listed in the following table.

LOT number	Part	Number	Length	Result
180906LP03	pSB1C3-K206000-34-AOX1	1-4	1675 bp	positive
180906LP06	pSB1C3-RNAG120-MlcRE-RE-BBa_E0840	5-8	1850 bp	negative
180906LP07	pSB1C3-oHybB-BBa_E0840	9-12	1479 bp	positive
180906LP08	pSB1C3-MlcRE	13-16	597 bp	positive
180906LP09	pSB1C3-SulA	17-20	901 bp	positive
180906LP10	pSB1C3-BBa_K02600-31-sulA	21-24	1040 bp	negative
180906LP11	pSB1C3-BBa_K02600-32-sulA	25-28	1040 bp	negative
180906LP12	pSB1C3-BBa_K02600-34-sulA	29-32	1040 bp	negative
180906LP13	pSB1C3-BBA_B0032-cspD	33-36	603 bp	negative

ONCs were prepared of the parts showing a positive result.

LOT number	Part
180907LP01	psB1C3-K206000-34-AOX1
180907LP02	psB1C3-oHybB-E0840
180907LP03	psB1C3-MlcRE
180907LP04	psB1C3-sulA

Restriction and ligation

The restriction was performed according to the following table. The preparations were incubated at first for 1 h at 37 °C and afterwards at 80 °C for 20 min.

Tube

1

2

3

4

5

6

7

8

9

10

11

12

Part

pSB1C3_RNAG120_MlcRE

pSB1C3-BBa-E0840

pSB1C3-BBa_K206000

31sulA

32sulA

34sulA

cspD

pSB1C3-BBa_B0032

cspD

34-AOX1

pSB1C3-BBa_I13453

pSB1C3

Lot#

180822BK02

180907NiF03

180829LD01

180905AW01

180905AW02

180905AW03

180905AW05

180514NB07

180905AW05

180906LP01

180822BK01

180906LP02

ρ

119 ng/µL

92 ng/µL

115 ng/µL

28 ng/µL

40 ng/µL

43 ng/µL

48 ng/µL

81 ng/µL

48 ng/µL

84 ng/µL

115 ng/µL

138 ng/µL

Use

vector

single insert

vector

single insert

vector

single insert

vector

single insert

target m

2000 ng

100 ng

2000 ng

100 ng

2000 ng

100 ng

2000 ng

100 ng

DNA

7.00 µL

16.00 µL

3.57 µL

2.50 µL

2.33 µL

2.08 µL

7.00 µL

2.08 µL

1.19 µL

17.34 µL

0.72 µL

NEBuffer 2.1

1.00 µL

2.00 µL

1.00 µL

3.00 µL

1.00 µL

EcoRI-HF

0.50 µL

XbaI

0.50 µL

SpeI

0.50 µL

PstI

0.50 µL

FastAP

0.50 µL

H2O

0.50 µL

4.43 µL

5.50 µL

5.67 µL

5.92 µL

0.50 µL

5.92 µL

6.81 µL

8.16 µL

7.28 µL

ad

10.00 µL

20.00 µL

10.00 µL

30.00 µL

10.00 µL

Länge Produkt

2697 bp

878 bp

2200 bp

565 bp

245 bp

2083 bp

245 bp

1200 bp

2200 bp

2070 bp

ρ final

83 ng/µL

64 ng/µL

92 ng/µL

10 ng/µL

56 ng/µL

10 ng/µL

67 ng/µL

10 ng/µL

After the gel extraction DNA concentrations were determined. Restriction 1 was not successful.

Restriction	Concentration [ng/µL]
2	3.8
3	8.3
8	5.0

The ligation was performed overnight according to the following table.

Tube		L1	L2	L3	L4	L5	L6	L7	L8
Product		pSB1C3-34-AOX1	pSB1C3-BBa_K206000-34-AOX1	pSB1C3_RNAG120_MlcRE-pSB1C3-BBa-E0840	pSB1C3-BBa_K206000-31sulA	pSB1C3-BBa_K206000-32sulA	pSB1C3-BBa_K206000-34sulA	pSB1C3-BBa_B0032-cspD	pSB1C3-cspD

Vector	Vector	pSB1C3	pSB1C3-BBa_K206000	pSB1C3_RNAG120_MlcRE	pSB1C3-BBa_K206000	pSB1C3-BBa_K206000	pSB1C3-BBa_K206000	pSB1C3-BBa_B0032	pSB1C3
	Tube	12	3	1	3	3	3	8	12
	ρ	10 ng/µL	8 ng/µL	83 ng/µL	8 ng/µL	8 ng/µL	8 ng/µL	5 ng/µL	10 ng/µL
	Länge	2070 bp	2200 bp	2697 bp	2200 bp	2200 bp	2200 bp	2083 bp	2070 bp
	V	1.47 µL	1.87 µL	0.28 µL	3.38 µL	3.38 µL	3.38 µL	9.19 µL	4.58 µL
Part 1	Part 1	34-AOX1	34-AOX1	pSB1C3-BBa-E0840	31sulA	32sulA	34sulA	cspD	cspD
	Tube	10	10	2	4	5	6	7	9
	ρ	10 ng/µL	10 ng/µL	64 ng/µL	10 ng/µL	10 ng/µL	10 ng/µL	10 ng/µL	10 ng/µL
	Länge	1200 bp	1200 bp	878 bp	565 bp	565 bp	565 bp	245 bp	245 bp
	V	8.53 µL	8.45 µL	1.19 µL	7.20 µL	7.20 µL	7.20 µL	5.40 µL	5.42 µL
Part 2	Part 2
	Tube
	ρ
	Länge
	V
T4 Buffer		2.00 µL	2.00 µL	1.00 µL	2.00 µL	2.00 µL	2.00 µL	2.00 µL	2.00 µL
T4 Ligase		0.50 µL	0.50 µL	0.50 µL	0.50 µL	0.50 µL	0.50 µL	0.50 µL	0.50 µL
H2O		7.00 µL	7.00 µL	7.02 µL	7.00 µL	7.00 µL	7.00 µL	2.90 µL	7.50 µL
ad		20.00 µL	20.00 µL	10.00 µL	20.00 µL	20.00 µL	20.00 µL	20.00 µL	20.00 µL

PCR for GG Assembly (+BjaIT) + RNAG120 + pGGA

PCR: 25 µL Q5 MM, 20 µL H₂O, 2.5 mL primer each, 0.25 µL template

Part	fw primer	rv primer	Template	LOT number	Conc [ng/µL]	pred. length
Bsa_HlyB	BsaI_HlyB_f	BsaI_HlyB_r	BBa_K554013	18090901	184	3800
Bsa_TolC	BsaI_TolC_f	BsaI_TolC_r	BBa_K554013	18090902	138	1300
Bsa_OmpT	BsaI_OmpT_f	BsaI_OmpT_r	180827LP01	18090903	177	1000
Bsa_pBAD	BsaI-dopAP_f	BsaI_pBAD_r	180829LD01	18090904	143	200
Bsa_NOTMlcRE	BsaI-dopAP_f	BsaI_RnaG_r	180822BK02	18090905	101	540
BjalT-OmpTSite-FLAG	BBPre_Syn_f	BBSuf_Syn_r	IDT(76546522)	18090906	94	300
RnaG120	RnaG120_f	RnaG120_r	180822BK02	18090907	76	120
pGGA	pGGA_Prep_f	pGGA_Prep_r	pGGA(NEBKit)	18090908	53	2170
RnaG120+Terminator	BBPre_Syn_f	RnaG120_r	180822BK02	18090909	77	314

1% agarose gel à all successful

PCR purification with thermos-kit à at -20 °C

GG Assembly after 180909 GGA

Transformation following the protocol by Zhang et al.

remaining ligation approaches (10 µL) were stored at -20 °C

180909DW01 psB1C3_BBa_B0034_AOX1

180909DW02 psB1C3_BBa_K206000_BBa_B0034_AOX1

180909DW03 psB1C3_BBa_K206000_BBa_B0031_SulA

180909DW04 psB1C3_BBa_K206000_BBa_B0032_SulA

180909DW05 psB1C3_BBa_K206000_BBa_B0034_SulA

180909DW06 psB1C3_BBa_B0032-cspD

180909DW07 pGGA_pBAD_HlyB-HlyD-TolC-OmpT

180909DW08pGGA_NotMlcRE-HlyB-HlyD-TolC-OmpT

GG Assembly

Procedure for the GGA:

PCR with Q5, with E. coli as a template or the existing parts
GGA where pGGA (Vector with 2174 bp), Assembly Mix und Buffer, DNA are used (fill with water). The DNA volume of the 3 to 7 inserts depends on the part’s length (in bp) and its density (in ng/µL)
Transformation, cPCR, mini, cryos, sequencing, characterization

Overview		Part 1	Part 2	Part 3	Part 4	Part 5	Part 6
Growth Inhbition	pBAD	cbtA	cspD	mraZ	sulA
Myristic Acid	pBAD	accA	accB	accC	accD	tesA
3MB	pBAD	ADH2	kivD	LeuA	LeuB	LeuC	LeuD
Toxin Synthesis	pBAD	BjaIT	HlyA
Excretion System	pBAD	HlyB	HlyD	TolC	OmpT

Calculation	length (bp)	density (ng/µL)	volume (µL)	Excretion
Promotor	161	143	0.2	pBAD
Part 1	3872	184	1.45194192	HlyB+HlyD
Part 2	1296	138	0.64797408	TolC
Part 3	975	177	0.38006954	ompT
Buffer			2
Mix			1
pGGA			1
H2O			13.3200145

Calculation	length (bp)	density (ng/µL)	volume (µL)	Excretion
Promotor	161	101	0.2	NotMlcRE
Part 1	3872	184	1.45194192	HlyB+HlyD
Part 2	1296	138	0.64797408	TolC
Part 3	975	177	0.38006954	ompT
Buffer			2
Mix			1
pGGA			1
H2O			13.3200145

Assembly protocol:

(37 °C, 1 min à 16 °C, 1min) 30 times

55 °C à 5 min

cPCR of the ligation from 08.09.18 and 09.09.18

Colonies have grown on all plates.

PCR preparation	1x	26x
10x Taq Buffer	2 µL	52 µL
dNTPs 10 mM	0.4 µL	10.4 µL
50 mM MgCl₂	0.6 µL	15.6 µL
fw primer	0.6 µL	15.6 µL
rv primer	0.6 µL	15.6 µL
Taq polymerase	0.1 µL	2.6 µL
H₂O ad 20 µL	14.5 µL	377 µL

The PCR programme was carried out as follows.

Step	Temperature	Time [s]
1	95 °C	600
2	95 °C	30
3	55 °C	30
4	72 °C	120
go to step 2 (30x)
5	72 °C	300

Control of the cPCR is shown in the following table and the following image.

Part	Number	Length	Gel
pSB1C3-BBa-K206000-BBa-B0032-sulA	1-3	1040 bp	negative
pSB1C3-BBa-K206000-BBa-B0034-sulA	4-6	1040 bp	negative
pSB1C3-BBa_B0032-cspD	7-9	603 bp	negative
pSB1C3-BBa_K206000-BBa_B0031-sulA	10-12	1040 bp	positive
pSB1C3-BBa_B0034-AOX1	13-15		negative
pGGA-NotMlcRE-HlyB-HlyD-TolC-OmpT	16-18		negative
pGGA-pBAD-HlyB-HlyD-TolC-OmpT	19-21	6304 bp	negative
pSB1C3-BBa-K206000-BBa-B0034-AOX1	22-24	1675 bp	positive

ONCs of the numbers 10 and 23 as well as from cryostock 180518BK06 were prepared.

The following samples were sent for sequencing.

LIGHTrun tube \| lab record Seq. ID 98ED24	180906NB04
LIGHTrun tube \| lab record Seq. ID 98ED24	180906NB03
LIGHTrun tube \| lab record Seq. ID 98ED24	180906LP02
LIGHTrun tube \| lab record Seq. ID 98ED24	180906NB05
LIGHTrun tube \| lab record Seq. ID 98ED24	180906NB06

Colony PCR of GGA (from the 9th of September)

A cPCR of two cell cultures (DH5α) with both: a) pGGA-NotMIcRE-HlyB-HlyD-TolC-OmpT and b) pGGA-pBAD-HlyB-HlyD-TolC-OmpT (from the 9^th of September) were prepared according to the table below to verify their presence. Due to the extreme length of the constructs, the insert was split in two using two primer pairs per construct.

PCR-mixture, for 9 colonies per plate and 2 PCR-preparations per colony (two different primer pairs)

		1x	Mastermix (38x) (2 preparations buffer)	MM primer pair 1	MM primer pair 2
10x DreamTaq Buffer		2 µL	76 µL
dNTPs 10 mM		0.4 µL	15.2 µL
50 mM MgCl₂		1.2 µL	45.6 µL
Primer 1	GGA_cPCR_fw	0.6 µL per primer (1 primer pair)	/	11.4 µL	/
Primer 1	BSAI_HlyB=rev		/	11.4 µL	/
Primer 2	BSAI_TolC_fw		/	/	11.4 µL
Primer 2	GGA_cPCR_rev		/	/	11.4 µL
DreamTaq Polymerase		0.1 µL	/	1.9 µL	1.9 µL
H₂O ad final volume (1x: 20 µL)		15.1 µL	573.8 µL	* ↓ 380 µL ↓ 18 x 20 µL	* ↓ 380 µL ↓ 18 x 20 µL

= 36 PCR mixtures in total

* (an arrow pointed from the 573.8 µL onto the marked fields saying “split between 2 master mixes (á 355.3 µL)”)

- 9 colonies have been picked per plate. Each colony has been dipped into the PCR mixture containing the first primer pair, and then into the mixture containing the second primer pair. The tips were then transferred into 1 mL of LB (+ CAMP) and incubated for 10 minutes at 37 °C.

(- The PCR program is described on the next page)

Restriction/Ligation

Part	pSB1C3_RNAG120_BBa-E0840		pSB1C3-BBa-E0840	BBa_J45503	pSB1C3-oHybB	34-AOX1
Lot #	180822BK02	180907NF03	180907NF03	180625NF12	180911LP03	180906LP01
C	119 ng/μL	92 ng/μL	92 ng/ μL	45 ng/ μL	69 ng/ μL	84 ng/ μL
Use	vector	vector	vector	single insert	vector	single insert
Target m	2000 ng		2000 ng	100 ng	2000 ng	100 ng
DNA	5.70 μL	21.81 μL	17.30 μL	2.24 μL	25.00 μL	4.80 μL
NEBuffer 2.1	1.00 μL	3.00 μL	3.00 μL	1.00 μL	3.00 μL	4.00 μL
EcoRI-HF	-	-	0.5 μL	0.5 μL	-	-
XbaI	-	0.5 μL	0.5 μL		-	0.5 μL
SpeI	0.5 μL	-	-	0.5 μL	0.5 μL
PstI	0.5 μL	0.5 μL	-	-	0.5 μL	0.5 μL
FastAP	0.5 μL	0.5 μL	0.5 μL		0.5 μL
H₂O	1.80 μL	3.69 μL	8.20 μL	5.76 μL	0.5 μL	30.20 μL
C_Final	68 ng/ μL	67 ng/ μL	10 ng/ μL	10 ng/ μL	10 ng/ μL	10 ng/ μL

Part	pSB1C3_K20600			31SulA	32SulA	34SulA	cspD	pSB1C3-BBa_B0032
Lot #	180829LD01		180905AW01		180905AW02	180905AW03	180905AW05	180514NB07
C	115 ng/μL		28 ng/μL		40 ng/ μL	43 ng/ μL	48 ng/ μL	81 ng/ μL
Use	vector		single insert		single insert	single insert	single insert	vector
Target m	2000 ng	100 ng			100 ng	100 ng	100 ng	2000 ng
DNA	4.88 μL		3.57 μL		2.5 μL	2.33 μL	2.08 μL	6.10 μL
NEBuffer 2.1	1.00 μL		1.00 μL		1.00 μL	1.00 μL	1.00 μL	1.00 μL
EcoRI-HF	-		-		-	-	-	-
XbaI	-		0.5 μL		0.5 μL	0.5 μL	0.5 μL	-
SpeI	0.5 μL		-		-	-	-	0.5 μL
PstI	0.5 μL		0.5 μL		0.5 μL	0.5 μL	0.5 μL	0.5 μL
FastAP	0.5 μL		-		-		-	0.5 μL
H₂O	2.62 μL		4.43 μL		5.5 μL	567 μL	5.92 μL	1.40 μL
C_Final	6 ng/ μL		10 ng/ μL		10 ng/ μL	10 ng/ μL	10 ng/ μL	10 ng/ μL

Part	pSB1C3	pSB1C3-I13453
Lot #	180906LP02	180822BK01
C	138 ng/μL	115 ng/μL
Use	vector	vector
Target m	2000 ng	2000 ng
DNA	14.49 μL	16.12 μL
NEBuffer 2.1	2.00 μL	2.00 μL
EcoRI-HF	-	-
XbaI	0.5 μL	-
SpeI	-	0.5 μL
PstI	0.5 μL	0.5 μL
FastAP	0.5 μL	0.5 μL
H₂O	2.01 μL	0.38 μL
C_Final	100 ng/ μL	7 ng/ μL

Ligations

Tube product	pSB1C3_RNAG120_MIcRE_BBa-E0840	pSB1C3-BBa-E0840_BBa_J45503	pSB1C3_oHybB-34-AOX1
vector	pSB1C3_RNAG120_MIcRe (68 ng/ μL, 0.35 μL)	pSB1C3-BBa-E0840 (10 ng/ μL, 4.29 μL)	pSB1C3_oHybB (10 ng/ μL, 1.55 μL)
Part 1	BBa-E0840 (67 ng/ μL, 1.15 μL)	BBa_J45503 (10 ng/ μL, 5.71 μL)	41-AOX1 (10 ng/ μL, 8.38 μL)
Part 2	-	-
T4 Buffer	1.00 μL	2.00 μL	2.00 μL
T4 Ligase	0.5 μL	0.5 μL	0.5 μL
H₂O	7.00 μL	7.5 μL	7.57 μL
ad	10.0 μL	20.00 μL	20.00 μL

Tube product	pSB1C3_mraz	pSB1C3-BBa- K20600-32SulA	pSB1C3-BBa- K20600-34SulA
vector	pSB1C3 (74 ng/ μL, 0.61 μL)	pSB1C3-BBa-k20600 (6 ng/ μL, 4.25 μL)	pSB1C3-BBa-k20600 (6 ng/ μL, 4.25 μL)
Part 1	mraz (10 ng/ μL, 10.0 μL)	32-SulA (10 ng/ μL, 7.20 μL)	34-SulA (10 ng/ μL, 7.20 μL)
Part 2	-	-
T4 Buffer	2.00 μL	2.00 μL	2.00 μL
T4 Ligase	0.5 μL	0.5 μL	0.5 μL
H₂O	6.89 μL	7.5 μL	5.63 μL
ad	20.0 μL	20.00 μL	20.00 μL

Tube product	pSB1C3_BBa-80032-cspD	pSB1C3-34-AOX1	pSB1C3-I13453-34-AOX1
vector	pSB1C3_BBa_80023 (8 ng/ μL, 5.74 μL)	pSB1C3 (100 ng/ μL, 0.15 μL)	pSB1C3-I13453 (7 ng/ μL, 42.21 μL)
Part 1	cspD (10 ng/ μL, 5.40 μL)	34-AOX1 (10 ng/ μL, 8.46 μL)	34-AOX1 (10 ng/ μL, 8.38 μL)
Part 2	-	-	-
T4 Buffer	2.00 μL	2.00 μL	2.00 μL
T4 Ligase	0.5 μL	0.5 μL	0.5 μL
H₂O	6.35 μL	6.89 μL	6.90 μL
ad	20.0 μL	20.00 μL	20.00 μL

Tube product	pSB1C3_oHybB-34-AOX1
vector	pSB1C3_oHybB (10 ng/ μL, 1.55 μL)
Part 1	34-AOX1 (10 ng/ μL, 8.38 μL)
Part 2	-
T4 Buffer	2.00 μL
T4 Ligase	0.5 μL
H₂O	7.57 μL
ad	20.0 μL

Gelextraction using GeneJetKit of Restrictions 3, 5, 9, 14, 16
Problems: 1 could not be detected on the gel, 2 did not show the right length on the gel

Colony PCR of the transformation performed on the 12^th of September

Mastermix

Substance	1x (volume in µl)	40x (volume in µl)
10x DreamTaq Buffer	2	80
dNTPs 10mM	0.4	16
MgCl₂ 50mM	1.2	48
Fw-Primer VF2	0.6	24
Rv-Primer VR	0.6	24
Taq-Polymerase	0.1	4
H₂O ad20µl	15.1	604

Colonies were picked with pipette-tips and transferred into 1 ml LB-Medium tubes after they were put in 20 µl aliquots of the mastermix of the following PCR reaction

PCR-Programm (30 cycles)

Temperature (°C)	Time (s)
95	180
95	30
55	30
72	120
72	600

180913 cPCR.PNG

The colonies of following bands were used to set up ONCs.

Band	Part	Length (bp)
6	pSB1C3-BBa_E0840-BBa_J45503	1571
10	pSB1C3-mraz	758
18	pSB1C3-BBa_K206000-BBa_B0032-sulA	995
31	pSB1C3-BBa_B0032-cspD	558

PCR of linear pSB1C3

Mastermix

Substance	1x (volume in µl)	3x (volume in µl)
5x HF Buffer	10	30
DNA-Template	0.5	1.5
F-Primer	0.35	1.05
R-Primer	0.35	1.05
Phusion-Polymerase	0.5	1.5
H₂O ad 50µl	37.3	111.9

PCR-Programm (35 cycles)

Temperature (°C)	Time (s)
98	120
98	30
55	30
72	180
72	600

The 3 set ups were purified with the “GeneJet PCR-Purification Kit” of Thermo Fisher and eluted in 100 µl ddH₂O.

C = 89 ng/µl

Restriction and ligation

Restriction

Part	pSB1C3-NOT-MlcRE	pSB1C3-BBa_Eo840	pSB1C3-oHybB	pSB1C3-K206000	pSB1C3	BBa_B0034-AOX1	BBa_B0034-sulA
DNA	38.46 µl	30.77 µl	22 µl	34.48 µl	22.47 µl	2.4 µl	2.33 µl
NEBBuffer 2.1	5 µl	4 µl	4 µl	4 µl	3 µl	2 µl	1 µl
EcoRI-HF	-	-	-	-	-	-	-
XbaI	-	0.5 µl	-	-	0.5 µl	0.5 µl	0.5 µl
SpeI	0.5 µl	-	0.5 µl	0.5 µl	-	-	-
PstI	0.5 µl	0.5 µl	0.5 µl	0.5 µl	0.5 µl	0.5 µl	0.5 µl
FastAP	0.5 µl	0.5 µl	0.5 µl	0.5 µl	0.5 µl	-	-
ddH₂O	5.04 µl	3.73 µl	5.51 µl	-	3.03 µl	14.6 µl	5.67 µl
ad	50 µl	40 µl	40 µl	40 µl	30 µl	20 µl	10 µl

The samples were incubated at 37°C for 1 h and following 20 min at 80°C. Samples 1-4 were applied on a 1% agarose gel and extracted through the “GeneJet Gel Extraction Kit” of Thermo Fisher.

Ligation

Product	pSB1C3-NOT-MlcRE-BBa_E0840	pSB1C3-oHybB-BBa_B0034-AOX1	pSB1C3-BBa_K206000-BBa_B0034-sulA	pSB1C3-BBa_B0034-AOX1
Vector	1.46 µl	0.44 µl	1.82 µl	0.24 µl
Part	8.5 µl	8.25 µl	10 µl	8.35 µl
T4 Buffer	2 µl	2 µl	2 µl	2 µl
T4 Ligase	0.5 µl	0.5 µl	0.5 µl	0.5 µl
ddH₂O	7.54 µl	8.81 µl	5.68 µl	8.91 µl
ad	20 µl	20 µl	20 µl	20 µl

The samples were incubated at 16°C overnight.

Template preparation for GGA by PCR

All PCR Reactions were prepared as written below.

8 µL	H₂O
0.2 µL	Template
1 µL	Fw-Primer
1 µL	Rv-Primer
10 µL	Q5 High-Fidelity 2X Master Mix

Tube	Part	Fw-Primer	Rv-Primer	Predicted length (bp)	Template
1	BsaI-pBAD	BsaI-pBAD-f	BsaI-pBAD-r	186	180909BK04
2	BsaI-NOTMlcRE	BsaI-NOTMlcRE-f	BsaI-NOTMlcRE-r	549	180909BK05
3	BsaI-HlyB	BsaI-HlyB-f	BsaI-HlyB-r	3895	180909BK01
4	BsaI-TolC	BsaI-TolC-f	BsaI-TolC-r	1322	180909BK02
5	BsaI-OmpT	BsaI-OmpT-f	BsaI-OmpT-r	1001	180909BK03
6	BsaI-ADH2	BsaI-ADH2-f	BsaI-ADH2-r	1100	180726LP03
7	BsaI-kivD	BsaI-kivD-f	BsaI-kivD-r	1686	180726LP01
8	BsaI-LeuA	BsaI-LeuA-f	BsaI-LeuA-r	1033	e. coli
9	BsaI-LeuB	BsaI-LeuB-f	BsaI-LeuB-r	1115	e. coli
10	BsaI-LeuC	BsaI-LeuC-f	BsaI-LeuC-r	1441	e. coli
11	BsaI-LeuD	BsaI-LeuD-f	BsaI-LeuD-r	664	e. coli
12	BsaI-accA	BsaI-accA-f	BsaI-accA-r	1017	e. coli
13	BsaI-accB	BsaI-accB-f	BsaI-accB-r	515	180703BK05
14	BsaI-accC	BsaI-accC-f	BsaI-accC-r	1384	180726LP04
15	BsaI-accD	BsaI-accD-f	BsaI-accD-r	951	IDT syn
16	BsaI-tesA	BsaI-tesA-f	BsaI-tesA-r	705	e. coli

PCR was performed using following Cycler-Protocol

Temperature (°C)	Time	Time (for e. coli templates)	Repeats
98	0:20	2:00	1
98	0:10	0:10	30
60	0:20	0:20	30
72	1:00	1:00	30
72	2:00	2:00	1
4	∞	∞	1

A gel electrophoresis was performed using a 1%-Agarose-Gel.

Only BsaI-ADH2 was successful.

Restriction and ligation of basic parts

Restriction was set up according to following table, performed at 37 °C for an hour and heat inactivated at 80 °C for 20 minutes.

	Tube	1	2	3	4	5	6
	Part	accA	accB	icsA	RnaG120	BjaIT-OmpTSite-FLAG	pSB1C3
	Lot#	180622NB05	180622NB06	180909BK07	180909BK09	180909BK08	180914LP01
	ρ	49 ng/µL	52 ng/µL	76 ng/µL	77 ng/µL	94 ng/µL	89 ng/µL
	Use
	target m

Volumes	DNA	2.04 µL	1.92 µL	1.32 µL	1.30 µL	1.06 µL	3.00 µL
	NEBuffer 2.1	1.00 µL	1.00 µL	1.00 µL	1.00 µL	1.00 µL	1.00 µL
	EcoRI-HF	0.50 µL	0.50 µL	0.50 µL	0.50 µL	0.50 µL	0.50 µL
	XbaI
	SpeI
	PstI	0.50 µL	0.50 µL	0.50 µL	0.50 µL	0.50 µL	0.50 µL
	FastAP
	H2O	5.96 µL	6.08 µL	6.68 µL	6.70 µL	6.94 µL	5.00 µL
	ad	10.00 µL	10.00 µL	10.00 µL	10.00 µL	10.00 µL	10.00 µL

	Länge Produkt

	ρ final	10 ng/µL	10 ng/µL	10 ng/µL	10 ng/µL	10 ng/µL	27 ng/µL

Ligation was set up according to following table and performed at 37 °C for an hour.

Tube		L1	L2	L3	L4	L5
Product		pSB1C3-accA	pSB1C3-accB	pSB1C3-icsA	pSB1C3-RnaG120	pSB1C3-BjaIT-OmpTSite-FLAG

Vector	Vector	pSB1C3	pSB1C3	pSB1C3	pSB1C3	pSB1C3
	Tube	6	6	6	6	6
	ρ	27 ng/µL	27 ng/µL	27 ng/µL	27 ng/µL	27 ng/µL
	Länge	2046 bp	2046 bp	2046 bp	2046 bp	2046 bp
	V	0.64 µL	1.09 µL	2.36 µL	1.48 µL	1.52 µL
Part 1	Part 1	accA	accB	icsA	RnaG120	BjaIT-OmpTSite-FLAG
	Tube	1	2	3	4	5
	ρ	10 ng/µL	10 ng/µL	10 ng/µL	10 ng/µL	10 ng/µL
	Länge	1000 bp	500 bp	120 bp	314 bp	300 bp
	V	7.30 µL	7.10 µL	3.70 µL	6.05 µL	5.95 µL
Part 2	Part 2
	Tube
	ρ
	Länge
	V
T4 Buffer		1.00 µL	1.00 µL	1.00 µL	1.00 µL	1.00 µL
T4 Ligase		0.50 µL	0.50 µL	0.50 µL	0.50 µL	0.50 µL
H2O		0.56 µL	0.32 µL	2.44 µL	0.97 µL	1.04 µL
ad		10.00 µL	10.00 µL	10.00 µL	10.00 µL	10.00 µL

Colony PCR of the transformation performed on the 9^th of September

Mastermix

Substance	1x (volume in µl)	20x (volume in µl)
10x DreamTaq Buffer	2	40
dNTPs 10mM	0.4	8
MgCl₂ 50mM	0.6	12
Fw-Primer VF2	0.6	12
Rv-Primer VR	0.6	12
Taq-Polymerase	0.1	2
ddH₂O ad 20µl	15.7	314

Colonies were picked with pipette-tips and transferred into 1 ml LB-Medium tubes after they were put in 20 µl aliquots of the mastermix of the following PCR reaction

PCR-Programm (30 cycles)

Temperature (°C)	Time (s)
95	180
95	30
55	30
72	360
72	600

Two ONCs of the part pSB1C3-NOT-MlcRE-HlyB-HlyD-TolC-OmpT were set up.

Colony PCR of the transformation performed on the 16^th of September

MasterMix

Substance	1x (volume in µl)	40x (volume in µl)
10x DreamTaq Buffer	2	80
dNTPs 10mM	0.4	16
MgCl₂ 50mM	0.6	24
Fw-Primer VF2	0.6	24
Rv-Primer VR	0.6	24
Taq-Polymerase	0.1	4
ddH₂O ad 20µl	15.7	628

Colonies were picked with pipette-tips and transferred into 1 ml LB-Medium tubes after they were put in 20 µl aliquots of the mastermix of the following PCR reaction

PCR-Program (30 cycles)

Temperature (°C)	Time (s)
95	180
95	30
55	30
72	360
72	600

The colonies of following bands were used to set up ONCs.

Band	Part
3	pSB1C3-accA
8	pSB1C3-accB
11	pSB1C3-icsA
16	pSB1C3-RnaG120
19	pSB1C3-BjaIT-OmpTSite-FLAG
25	pSB1C3-oHybB-BBa_B0034-AOX1

Test digestion of the sequencing parts

Tube	Part	Sequencing ID
1	180918AK09	47GJ15
2	180918AK10	47GJ16
3	180918AK11	47GJ18
4	180918AK12	47GJ19
5	180918AK13	47GJ20
6	180918AK14	47GJ22
7	180917K03	-
8	180917K04	-
9	180726LP01	47GJ23
10	180726LP02	empty
11	180726LP03	47GJ27
12	180726LP04	-
13	180803NB02	47GJ29
14	180914AW01	47GJ31
15	180914AW02	47GJ32

Master mix for 16 samples:

2 µL DNA
2 µL EcoR1
2 µL FastAP
16 µL NEB 2.1
96 µL H₂O

Incubated the samples for 1 h at 37°C.

Restriction

	Tube	1	2	3	4	5	6	7	8
	Part	pSB1C3_RNAG120_MlcRE	pSB1C3-E0240	pSB1C3-E0240	BBa_J45503	pSB1C3-oHybB	34-AOX1	pSB1C3-I13453	BBa_B0032_cbtA
	Lot#	180914AW05	180817LD10	180817LD10	180625NiF12	180915BK01	180906LP01	180914AW06	180731NF04
	ρ	118 ng/µL	200 ng/µL	200 ng/µL	45 ng/µL	170 ng/µL	84 ng/µL	115 ng/µL	173 ng/µL
	Use	vector	vector	vector	single insert	vector	single insert	vector	vector
	target m	2000 ng	2000 ng	2000 ng	100 ng	2000 ng	100 ng	2000 ng	2000 ng

Volumes	DNA	6.77 µL	10.00 µL	6.40 µL	2.24 µL	18.00 µL	4.80 µL	16.12 µL	11.56 µL
	NEBuffer 2.1	1.00 µL	2.00 µL	1.00 µL	1.00 µL	3.00 µL	4.00 µL	2.00 µL	2.00 µL
	EcoRI-HF			0.50 µL	0.50 µL
	XbaI		0.50 µL	0.50 µL			0.50 µL		0.50 µL
	SpeI	0.50 µL			0.50 µL	0.50 µL		0.50 µL
	PstI	0.50 µL	0.50 µL			0.50 µL	0.50 µL	0.50 µL	0.50 µL
	FastAP	0.50 µL	0.50 µL	0.50 µL		0.50 µL		0.50 µL	0.50 µL
	H2O	1.80 µL	6.50 µL	1.10 µL	5.76 µL	7.50 µL	30.20 µL	0.38 µL	4.94 µL
	ad	10.00 µL	20.00 µL	10.00 µL	10.00 µL	30.00 µL	40.00 µL	20.00 µL	20.00 µL

	Länge Produkt					2200 bp		2200	394

	ρ final	10 ng/µL	14 ng/µL	10 ng/µL	10 ng/µL	102 ng/µL	10 ng/µL	17 ng/µL	8 ng/µL

Ligation

Tube		L1	L2	L3
Product		pSB1C3_RNAG120_MlcRE-pSB1C3-E0240	pSB1C3-I13453-34-AOX1	pSB1C3-I13453-BBa_B0032_cbtA

Vector	Vector	pSB1C3_RNAG120_MlcRE	pSB1C3-I13453	pSB1C3-I13453
	Tube	1	7	7
	ρ	10 ng/µL	17 ng/µL	17 ng/µL
	Länge	2697 bp	2200 bp	2200 bp
	V	4.70 µL	0.91 µL	2.11 µL
Part 1	Part 1	pSB1C3-E0240	34-AOX1	BBa_B0032_cbtA
	Tube	2	6	8
	ρ	14 ng/µL	10 ng/µL	8 ng/µL
	Länge	878 bp	1200 bp	394 bp
	V	10.92 µL	8.38 µL	8.02 µL
Part 2	Part 2
	Tube
	ρ
	Länge
	V
T4 Buffer		2.00 µL	2.00 µL	2.00 µL
T4 Ligase		0.50 µL	0.50 µL	0.50 µL
H2O		1.88 µL	8.21 µL	7.37 µL
ad		20.00 µL	20.00 µL	20.00 µL

PCR of several parts

Mastermix for BBa_B0031-sulA and BBa_B0034-sulA

Substance	2x (volume in µl)
5x Phusion HF Buffer	20
dNTPs 10mM	2
F-Primer	0.7
R-Primer	0.7
Phusion-Polymerase	4
DNA-Template	2
ddH₂O ad 100µl	70.6

PCR-Program (30 cycles)

Temperature (°C)	Time (s)
98	180
98	10
55	30
72	60
72	300

Mastermix for pSB1C3

Substance	1x (volume in µl)
5x Phusion HF Buffer	10
dNTPs 10mM	1
F-Primer	0.35
R-Primer	0.35
Phusion-Polymerase	0.5
DNA-Template	0.5
ddH₂O ad 50µl	37.3

PCR-Program (35 cycles)

Temperature (°C)	Time (s)
98	120
98	30
55	30
72	180
72	600

PCR-Products were applied to a 1% agarose gel. PSB1C3 was cut out and purified by using the “GeneJet Gelextraction Kit” by Thermo Fisher.

https://static.igem.org/mediawiki/2018/2/24/T--Hamburg--180924_PCR.JPG

Restriction, ligation and transformation

Restriction

Tube	1	2	5	6	9	12	16	17
Part	pSB1C3_RNAG120_MlcRE	pSB1C3-E0240	pSB1C3-oHybB	34-AOX1	pSB1C3-BBa_K206000	34sulA	pSB1C3-I13453	pSB1C3-BBa_B0032_cbtA
Lot#	180921OM02	180921OM01	180921OM03	180920LP01	180829LD02	180905AW03	180914AW06	180731NF04
ρ	113 ng/µL	123	186 ng/µL	575 ng/µL	50 ng/µL	43 ng/µL	67 ng/µL	173.5
Use	vector	vector	vector	single insert	vector	single insert	vector	vector
target m	2000 ng	2000 ng	2000 ng	100 ng	2000 ng	100 ng	2000 ng	2000 ng

DNA	17.70 µL	16.26 µL	10.75 µL	0.17 µL	40.00 µL	2.33 µL	29.85 µL	11.53 µL
NEBuffer 2.1	3.00 µL	2.00 µL	2.00 µL	4.00 µL	5.00 µL	1.00 µL	4.00 µL	2.00 µL
EcoRI-HF
XbaI		0.50 µL		0.50 µL		0.50 µL		0.50 µL
SpeI	0.50 µL		0.50 µL		0.50 µL		0.50 µL
PstI	0.50 µL	0.50 µL	0.50 µL	0.50 µL	0.50 µL	0.50 µL	0.50 µL	0.50 µL
FastAP	0.50 µL	0.50 µL	0.50 µL		0.50 µL		0.50 µL	0.50 µL
H2O	7.80 µL	0.24 µL	5.75 µL	34.83 µL	3.50 µL	5.67 µL	4.65 µL	4.97 µL
ad	30.00 µL	20.00 µL	20.00 µL	40.00 µL	50.00 µL	10.00 µL	40.00 µL	20.00 µL

Länge Produkt			2200 bp				2200	394

ρ final	67 ng/µL	100 ng/µL	10 ng/µL	3 ng/µL	6 ng/µL	10 ng/µL	23 ng/µL	100 ng/µL

Probes were incubated for 1h at 37°C and following 20 min at 80°C.

Restriction 1.2,3.9,16 and 17 were applied to a 1% agarose gel and purified by using the “GeneJet Gel Extraction Kit” from Thermo Fisher.

Restriction 2 and 17 didn’t work.

Ligation

Product		pSB1C3_RNAG120_MlcRE-pSB1C3-E0240	pSB1C3-BBa_K206000-34sulA	pSB1C3-I13453-34-AOX1

Vector	Vector	pSB1C3_RNAG120_MlcRE	pSB1C3-BBa_K206000	pSB1C3-I13453
	Tube	1	9	16
	ρ	67 ng/µL	6 ng/µL	23 ng/µL
	Länge	2697 bp	2200 bp	2200 bp
	V	0.35 µL	4.25 µL	0.67 µL
Part 1	Part 1	pSB1C3-E0240	34sulA	34-AOX1
	Tube	2	12	6
	ρ	100 ng/µL	10 ng/µL	3 ng/µL
	Länge	878 bp	565 bp	1200 bp
	V	0.77 µL	7.20 µL	33.80 µL
Part 2	Part 2
	Tube
	ρ
	Länge
	V
T4 Buffer		1.00 µL	2.00 µL	4.00 µL
T4 Ligase		0.50 µL	0.50 µL	0.50 µL
H2O		7.38 µL	5.63 µL	1.02 µL
ad		10.00 µL	20.00 µL	40.00 µL

Probes were incubated at 37°C for 1h.

The ligations were transformed into DH5α.

150 µl Competent cell aliquots were thawed on ice for 15-30 min.
10 µl DNA were added and mixed
Cells were incubated for 35 min on ice
Heat-shock: 42°C for 45 sec, then put back on ice for 2-5 min
Pre-warm 850 µl LB-Medium
Add pre-warmed LB-Medium into the competent cells. 37°C 900 rpm 1 h
Cetrifuge 4500 rpm 2 min to collect cells.
Put cells on appropriate plates or medium
Incubation at 37°C overnight.

Lysis and protein assay of BjaIT

The stored pellet of the 21^st of September was lysed.

Pellets were resuspended in 50 µl PBS & incubated for 5 min on ice
Lysate was sonified in a waterbath for 30 sec
Incubation on ice for 15 min
Zentrifugation 3 min; 11000rpm; 11°C

Bradford-Assay:

99 µl ddH₂O + 1 µl lysate were mixed with 200 µl Bradford-Reagent
OD-measurement at 595 nm

A linear regression was made by using BSA

790 µl H₂O + 10 µl BSA

780 µl H₂O + 20 µl BSA

796 µl H₂O + 4 µl BSA

792 µl H₂O + 8 µl BSA

784 µl H₂O + 16 µl BSA

concentration [µg/µL]	Extinction linear regression	measured extinction of the probes (induced)	calculated concentration [µg/µL]
1	0.079	0.062	0.06963788301
2	0.126	0.067	0.208913649
4	0.24
8	0.332	measured extinction of the probes (not induced)
16	0.363	0.055	-0.1253481894
		0.053	-0.1810584958

			y = 0.0359x + 0.0595

PCR of the missing parts and the contaminated GGA PCR products

Template	Part	DNA-concentration [ng/µL]	Lot number
180921BK05	accC (Bsa1)	13.8	180925BK01
180921BK06	accD (Bsa1)	7.4	180925BK02
180921BK10	ADH2 (Bsa1)	14.9	180925BK03
180921BK11	kivD (Bsa1)	13	180925BK04
180921BK16	pBAD (Bsa1)	118.73	180925BK05
180921BK17	HlybB (Bsa1)	12.84	180925BK06
E.coli	LeuD (Bsa1)	-	-

PCR program:

		For leuD
98°C	20 s	98°C	3 min
98°C	10 s	98°C	10 s
60°C	20 s	52°C	20 s
72°C	2 min	72°C	30 s
Go to step 2 30x
72°C	2 min	72°C	3 min

Restriction

Tube

1

2

3

4

5

6

7

8

9

10

11

12

13

14

15

16

17

Part

pSB1C3_RNAG120_MlcRE

pSB1C3-E0240

BBa_J45503

pSB1C3-oHybB

34-AOX1

pSB1C3

mraz

pSB1C3-BBa_K206000

31sulA

32sulA

34sulA

cspD

pSB1C3-BBa_B0032

pSB1C3

pSB1C3-I13453

pSB1C3-BBa_B0032_cbtA

Lot#

180921OM02

180921OM01

180817LD10

180625NiF12

180921OM03

180920LP01

180906LP02

180622NB08

neue Miniprep

180905AW03

neue Miniprep

180924AW1

180925NaF04

180731NF04

ρ

113 ng/µL

123

200 ng/µL

45 ng/µL

186 ng/µL

575 ng/µL

138 ng/µL

44.175

50 ng/µL

28 ng/µL

40 ng/µL

43 ng/µL

48 ng/µL

81 ng/µL

95 ng/µL

126.15

173.5

Use

vector

single insert

vector

single insert

vector

single insert

vector

single insert

vector

target m

2000 ng

100 ng

2000 ng

100 ng

2000 ng

100 ng

2000 ng

100 ng

2000 ng

Volumes

DNA

17.70 µL

16.26 µL

10.00 µL

2.24 µL

10.75 µL

0.17 µL

14.49 µL

2.26 µL

40.00 µL

3.57 µL

2.50 µL

2.33 µL

2.08 µL

24.84 µL

21.15 µL

15.85 µL

11.53 µL

NEBuffer 2.1

3.00 µL

2.00 µL

1.00 µL

2.00 µL

4.00 µL

2.00 µL

1.00 µL

5.00 µL

1.00 µL

3.00 µL

2.00 µL

EcoRI-HF

0.50 µL

XbaI

0.50 µL

SpeI

0.50 µL

PstI

0.50 µL

FastAP

0.50 µL

H2O

7.80 µL

0.24 µL

6.50 µL

5.76 µL

5.75 µL

34.83 µL

2.01 µL

5.74 µL

3.50 µL

4.43 µL

5.50 µL

5.67 µL

5.92 µL

0.66 µL

4.35 µL

0.65 µL

4.97 µL

ad

30.00 µL

20.00 µL

10.00 µL

20.00 µL

40.00 µL

20.00 µL

10.00 µL

50.00 µL

10.00 µL

30.00 µL

20.00 µL

Länge Produkt

2200 bp

2200

394

ρ final

67 ng/µL

100 ng/µL

10 ng/µL

3 ng/µL

100 ng/µL

10 ng/µL

6 ng/µL

10 ng/µL

8 ng/µL

67 ng/µL

7 ng/µL

100 ng/µL

Ligation

Tube

L1

L2

L3

L4

L5

L6

L7

L8

L9

L10

L11

Product

pSB1C3_RNAG120_MlcRE-pSB1C3-E0240

pSB1C3-E0240-BBa_J45503

pSB1C3-oHybB-34-AOX1

pSB1C3-mraz

pSB1C3-BBa_K206000-32sulA

pSB1C3-BBa_K206000-34sulA

pSB1C3-BBa_B0032-cspD

pSB1C3-34-AOX1

pSB1C3-I13453-34-AOX1

pSB1C3-I13453-pSB1C3-BBa_B0032_cbtA

pSB1C3-oHybB-pSB1C3-E0240

Vector

Vector

pSB1C3_RNAG120_MlcRE

pSB1C3-E0240

pSB1C3-oHybB

pSB1C3

pSB1C3-BBa_K206000

pSB1C3-BBa_B0032

pSB1C3

pSB1C3-I13453

pSB1C3-oHybB

Tube

1

3

5

7

9

14

15

16

5

ρ

67 ng/µL

10 ng/µL

100 ng/µL

6 ng/µL

8 ng/µL

67 ng/µL

7 ng/µL

10 ng/µL

Länge

2697 bp

2948 bp

2200 bp

2070 bp

2200 bp

2083 bp

2070 bp

2200 bp

V

0.35 µL

4.29 µL

1.55 µL

0.61 µL

4.25 µL

5.74 µL

0.22 µL

2.21 µL

5.12 µL

2.00 µL

Part 1

Part 1

pSB1C3-E0240

BBa_J45503

34-AOX1

mraz

32sulA

34sulA

cspD

34-AOX1

pSB1C3-BBa_B0032_cbtA

pSB1C3-E0240

Tube

2

4

6

8

11

12

13

6

17

2

ρ

100 ng/µL

10 ng/µL

3 ng/µL

10 ng/µL

3 ng/µL

100 ng/µL

Länge

878 bp

393 bp

1200 bp

458 bp

565 bp

245 bp

1200 bp

394 bp

878 bp

V

0.77 µL

5.71 µL

33.80 µL

10.00 µL

7.20 µL

5.40 µL

34.12 µL

33.80 µL

0.64 µL

0.80 µL

Part 2

Part 2

Tube

ρ

Länge

V

T4 Buffer

1.00 µL

2.00 µL

4.00 µL

2.00 µL

4.00 µL

5.00 µL

1.00 µL

T4 Ligase

0.50 µL

H2O

7.38 µL

7.50 µL

0.15 µL

6.89 µL

5.63 µL

6.35 µL

1.16 µL

8.48 µL

2.74 µL

5.70 µL

ad

10.00 µL

20.00 µL

40.00 µL

20.00 µL

40.00 µL

50.00 µL

10.00 µL

Title: cPCR of transformation (24.09.18) and pSB1C3-MIcRE (plated out again)

Mix for 20 approaches of PCR:

Taq-Buffer	40 μL
dNTPs	8 μL
MgCl₂	12 μL
VP2	12 μL
VR	12 μL
Taq-Polymerase	8 μL
H₂O	316 μL

PCR-program:

temperature	time
95 °C	3:00 Min
95 °C	0:30 Min
55 °C	0:30 Min
72 °C	2:00 Min
72 °C	5:00 Min
4 °C	∞

clones 1-8: pSB1C3-BBa_K206000-BBa_0034-SulA (L6)

clones 9-16: pSB1C3-MIcRE (derived from 180618BK01)

clones 17-19: pSB1C3-I13453-BBa_0034-AOX1 (L9)

Overnight culture of clone 14 (pSB1C3-MIcRE) (all other clones showed not estimated bands on gel)

SDS-PAGE of ldhA

Dilute 10x running buffer until 1x concentration is reached then pouring into run chamber.

Be aware of installing glas plates opposite of the gel to prevent run buffer from escaping the chamber!

Loadingscheme of gel:

DH5α			Page Ruler	ldhA + arabinose (+)			ldhA + arabinose (-)
2 μL	10 μL	20 μL	5 μL	2 μL	10 μL	20 μL	2 μL	10 μL	20 μL

Gel was runned für 120 minutes at 80 V. Gathering gel was removed. Seperation gel was washed for 2 minutes with ddH₂O and afterwards incubated for 20 minutes in 15 mL fixing solution. The gel was washed twice with ddH₂O. Incubation of the gel for 20 minutes in 15 mL coloring solution.

(gel lost)

Pouring of PAA-gels (15 %)

	seperation gel	gathering gel
acryl amide	6.75 mL	750 μL
seperation gel buffer	4.5 mL	-
gathering gel buffer	-	1.7 mL
H₂O	6.75 mL	4.25 mL
10 % APS	60 μL	20 μL
TEMED	13.5 μL	12 μL

Repetition of the PCR for generating LeuD

Two different protocols were used.

Mastermix 1

Substance	1x volume (µl)
2x Q5 Mastermix	12.5
F-Primer	0.2
R-Primer	0.2
DNA-Template (DH5α)	1

Mastermix 2

Substance	1x volume (µl)
5x Phusion buffer	4
F-Primer	0.2
R-Primer	0.2
10 mM dNTPs	0.4
Phusion polymerase	0.2
DNA-Template (DH5α)	1

PCR-program (30 cycles)

Temperature (°C)	Time (s)
98	120
98	10
55	20
72	30
72	300

Both PCR-products were applied to an 1% agarose gel. The PCR using Mastermix 1 didn’t work therefore just one product was cut up and purified using the “GenJet Gel Extraction Kit” from Thermo Fisher.

https://static.igem.org/mediawiki/2018/c/ce/T--Hamburg--180926.jpg

GGA-Assembly

Calculation	Länge (bp)	Dichte (ng/µL)	Volumen (µL)	3MB
Promotor	161	66	0.2	pBAD
Part 1	1050	14.9	3.6	ADH2
Part 2	1650	13	5.5	kivD
Part 3	1590	40	2.4	LeuA
Part 4	1100	44	1.7	LeuB
Part 5	1410	47	2	LeuC
Part 6	630	29.3	1.4	LeuD
Buffer			2
Mix			1
pGGA			3
H2O			0



Calculation	Länge (bp)	Dichte (ng/µL)	Volumen (µL)	Excretion
Promotor	161	66	0.2	pBAD
Part 1	3872	12.84	13	HlyB+HlyD
Part 2	1296	186	0.4807549633	TolC
Part 3	975	105	0.6406886582	ompT
Buffer			2
Mix			1
pGGA			3
H2O			0

Calculation	Länge (bp)	Dichte (ng/µL)	Volumen (µL)	Myristic Acid
Promotor	161	66	0.2	pBAD
Part 1	993	163	0.4203328799	accA
Part 2	489	204	0.1653904432	accB
Part 3	1358	170	0.5511661886	accC
Part 4	925	209	0.3053705603	accD
Part 5	679	74	0.6330962978	tesA
Buffer			2
Mix			1
pGGA			3
H2O			11.72464363

Calculation	Länge (bp)	Dichte (ng/µL)	Volumen (µL)	Growth Inhibition
Promotor	161	66	0.2	pBAD
Part 1	410	58.154	0.4864475091	34cbtA
Part 2	260	65.224	0.2750411264	34cspD
Part 3	500	66.294	0.5203882713	34mraZ
Part 4	565	87.334	0.4463718673	34sulA
Buffer			2
Mix			1
pGGA			1
H2O			14.07175123

Restriction, ligation and transformation

Restriction

Tube	2	17
Part	pSB1C3-E0240	pSB1C3-BBa_B0032_cbtA
Lot#	180925NaF02	180926NaF03
ρ	144.673	93.011
Use	vector	vector
target m	1000 ng	1000 ng

DNA	6.91 µL	10.75 µL
NEBuffer 2.1	1.00 µL	2.00 µL
EcoRI-HF
XbaI	0.50 µL	0.50 µL
SpeI
PstI	0.50 µL	0.50 µL
FastAP
H2O	1.09 µL	6.25 µL
ad	10.00 µL	20.00 µL

Länge Produkt	878 bp	394

ρ final	11 ng/µL	7 ng/µL

The probes were incubated at 37°C for 1h following another 20 min at 80°C. The products were then applied to a 1% agarose gel and purified using the “GenJet Gel Extraction Kit” from Thermo Fisher.

pSB1C3_RNAG120_MlcRE, pSB1C3-oHybB, 34-AOX1, pSB1C3-I13453 were already digested on September 25.

Ligation

Tube		L1	L9	L10	L11
Product		pSB1C3_RNAG120_MlcRE-pSB1C3-E0240	pSB1C3-I13453-34-AOX1	pSB1C3-I13453-pSB1C3-BBa_B0032_cbtA	pSB1C3-oHybB-pSB1C3-E0240

Vector	Vector	pSB1C3_RNAG120_MlcRE	pSB1C3-I13453	pSB1C3-I13453	pSB1C3-oHybB
	Tube	1	16	16	5
	ρ	18 ng/µL	28 ng/µL	28 ng/µL	26 ng/µL
	Länge	2697 bp	2200 bp	2200 bp	2200 bp
	V	1.32 µL	0.55 µL	1.27 µL	0.76 µL
Part 1	Part 1	pSB1C3-E0240	34-AOX1	pSB1C3-BBa_B0032_cbtA	pSB1C3-E0240
	Tube	2	6	17	2
	ρ	11 ng/µL	3 ng/µL	7 ng/µL	11 ng/µL
	Länge	878 bp	1200 bp	394 bp	878 bp
	V	6.71 µL	33.80 µL	8.79 µL	7.01 µL
Part 2	Part 2
	Tube
	ρ
	Länge
	V
T4 Buffer		1.00 µL	4.00 µL	2.00 µL	1.00 µL
T4 Ligase		0.50 µL	0.50 µL	0.50 µL	0.50 µL
H2O		0.47 µL	1.15 µL	7.44 µL	0.72 µL
ad		10.00 µL	40.00 µL	20.00 µL	10.00 µL

The ligations were incubated at 37°C for 1 h.

The ligations were transformed into DH5α.

150 µl Competent cell aliquots were thawed on ice for 15-30 min.
10 µl DNA were added and mixed
Cells were incubated for 35 min on ice
Heat-shock: 42°C for 45 sec, then put back on ice for 2-5 min
Pre-warm 850 µl LB-Medium
Add pre-warmed LB-Medium into the competent cells. 37°C 900 rpm 1 h
Cetrifuge 4500 rpm 2 min to collect cells.
Put cells on appropriate plates or medium
Incubation at 37°C overnight.

Proteinconcentration by Bradford

µg/µL	Extinktion 1	Extinktion 2	Mean
1	0.074	0.055	0.0645		DH5a	ldha -	ldha +	bjaIT -	bjaIT +
2	0.113	0.115	0.114	Extinktion 1	0.098	0.08	0.11	0.098	0.171
4	0.203	0.212	0.2075	Extinktion 2	0.103	0.076	0.102	0.122	0.151
8	0.285	0.277	0.281	Mean	0.1005	0.078	0.106	0.11	0.161
16	0.318	0.305	0.3115


					Mean	Konzentration [µg/µL]	Input 1400 µg Protein	ad 1100 µL H2O	Konzentration Stamm[µg/µL]
				DH5a	0.1005	1.879	745.274	354.726	1.273
				ldha+	0.106	1.981	706.604	393.396	1.273
				ldha-	0.078	1.458	960.256	139.744	1.273
				bjaIT+	0.161	3.009	465.217	634.783	1.273
				bjaIT-	0.11	2.056	680.909	419.091	1.273

					Input 20µg	Input 30	Input 40 µg
				Ansatz	15.71	23.57	31.43
				Wasser	15.71	7.86	0.00

The standard curve set up analog to 180924. After the measurement of the concentration, the samples were adjusted on 1400 µg protein. We added 5x SDS-Loading Dye and incubated for 10 min at 95°C. The samples used for a Western Blot and stored at -20°C.

cPCR of 3A Assembly

Mastermix	1x	40x	10x for GGA
10x Dream Taq buffer	2 µL	80 µL	20 µL
dNTPs (10 mM)	0.4 µL	16 µL	8 µL
MgCl₂(50 mM)	1.2 µL	48 µL	6 µL
Fw Primer VF2	0.6 µL	24 µL	6 µL (GGA_cPCR_f)
Rv Primer vR	0.6 µL	24 µL	6 µL (GGA_cPCR_r)
Taq Polymerase	0.1 µL	4 µL	1 µL
H₂O_dest	15.1 µL	604 µL	151 µL

Ligation	Part	Tube
L1	pSB1C3-RNAG120_MlcRE-pSB1C3-E0240	1-4
L9	pSB1C3-I13453-34-AOX1	5-12
L10	pSB1C3-I13453-BBa_B0032_cbtA	13-25
L11	pSB1C3-oHybB-E0240	26-38

PCR-Program:

95°C	3 min
95°C	30 s
55°C	30 s
72°C	1:45 min
Go to step 2 35x
72°C	5 min

GGA Program:

95°C	3 min
94°C	30 s
55°C	30 s
72°C	7 min
Go to step 2 30x
72°C	10 min

Restriktion

Tube	5	6	7	13	19	20	21	22
Part	pSB1C3-oHybB	34-AOX1	pSB1C3	cspD	accD	ldhA	ompT	RNAG120
Lot#	180921OM03	180920LP01	180914LP01	180622NB04	180921BK09	180622NB01	180827LP01	180909BK09
ρ	186 ng/µL	575 ng/µL	89 ng/µL	50 ng/µL	213 ng/µL	57 ng/µL	68 ng/µL	77 ng/µL
Use	vector	single insert	vector	single insert	single insert	single insert	single insert	single insert
target m	1000 ng	100 ng	1000 ng	100 ng	100 ng	100 ng	100 ng	100 ng

DNA	5.38 µL	0.17 µL	11.24 µL	2.00 µL	0.47 µL	1.77 µL	1.47 µL	1.30 µL
NEBuffer 2.1	1.00 µL	4.00 µL	2.00 µL	1.00 µL	1.00 µL	1.00 µL	1.00 µL	1.00 µL
EcoRI-HF			0.50 µL	0.50 µL	0.50 µL	0.50 µL	0.50 µL	0.50 µL
XbaI		0.50 µL
SpeI	0.50 µL
PstI	0.50 µL	0.50 µL	0.50 µL	0.50 µL	0.50 µL	0.50 µL	0.50 µL	0.50 µL
FastAP	0.50 µL		0.50 µL
H2O	2.12 µL	34.83 µL	5.26 µL	6.00 µL	7.53 µL	6.23 µL	6.53 µL	6.70 µL
ad	10.00 µL	40.00 µL	20.00 µL	10.00 µL	10.00 µL	10.00 µL	10.00 µL	10.00 µL

Länge Produkt	2200 bp	1200 bp	2070	222	915	987	954	120

ρ final	10 ng/µL	3 ng/µL	50 ng/µL	10 ng/µL	10 ng/µL	10 ng/µL	10 ng/µL	10 ng/µL

The probes were incubated at 37°C for 1h following another 20 min at 80°C. Probe 1 and 2 were applied to a 1% agarose gel and purified using the “GeneJet Gel Extraction Kit” from Thermo Fisher.

Since the concentrations were very low no ligations were possible.

Probes were stored at 4°C overnight.

Restriction, ligation and transformation

Restriction

Tube	1	2	6	7	13	16	17	19	20	21	22
Part	pSB1C3_RNAG120_MlcRE	pSB1C3-E0240	34-AOX1	pSB1C3	cspD	pSB1C3-I13453	pSB1C3-BBa_B0032_cbtA	accD	ldhA	ompT	RNAG120
Lot#	180925NaF01	180925NaF02	180920LP01	180914LP01	180622NB04	180925NaF04	180926NaF03	180921BK09	180622NB01	180827LP01	180909BK09
ρ	140 ng/µL	144.673	575 ng/µL	89 ng/µL	50 ng/µL	126 ng/µL	93.011	213 ng/µL	57 ng/µL	68 ng/µL	77 ng/µL
Use	Vector	vector	single insert	vector	single insert	vector	vector	single insert	single insert	single insert	single insert
target m	1000 ng	1000 ng	100 ng	1000 ng	100 ng	1000 ng	1000 ng	100 ng	100 ng	100 ng	100 ng

DNA	7.15 µL	6.91 µL	0.17 µL	11.24 µL	2.00 µL	7.93 µL	10.75 µL	0.47 µL	1.77 µL	1.47 µL	1.30 µL
NEBuffer 2.1	1.00 µL	1.00 µL	4.00 µL	2.00 µL	1.00 µL	2.00 µL	2.00 µL	1.00 µL	1.00 µL	1.00 µL	1.00 µL
EcoRI-HF				0.50 µL	0.50 µL			0.50 µL	0.50 µL	0.50 µL	0.50 µL
XbaI		0.50 µL	0.50 µL				0.50 µL
SpeI	0.50 µL					0.50 µL
PstI	0.50 µL	0.50 µL	0.50 µL	0.50 µL	0.50 µL	0.50 µL	0.50 µL	0.50 µL	0.50 µL	0.50 µL	0.50 µL
FastAP	0.50 µL			0.50 µL		0.50 µL
H2O	0.35 µL	1.09 µL	34.83 µL	5.26 µL	6.00 µL	8.57 µL	6.25 µL	7.53 µL	6.23 µL	6.53 µL	6.70 µL
ad	10.00 µL	10.00 µL	40.00 µL	20.00 µL	10.00 µL	20.00 µL	20.00 µL	10.00 µL	10.00 µL	10.00 µL	10.00 µL

Länge Produkt	2697 bp	878 bp	1200 bp	2070	222	2200	394	915	987	954	120

ρ final	100 ng/µL	8 ng/µL	3 ng/µL	19 ng/µL	10 ng/µL	11 ng/µL	5 ng/µL	10 ng/µL	10 ng/µL	10 ng/µL	10 ng/µL

The probes were incubated for 1h at 37°C following another 20 min at 80°C.

Restriction 1.2,7.16 and 17 were applied to a 1% agarose gel and purified using the “GeneJet Gel Extraction Kit” of Thermo Fisher.

PSB1C3-oHybB had been digested on September 14 and was kept and therefor used for the following ligations. The digested parts of September 29 were used as well.

Ligation

Tube		L9	L10	L11	L13	L14	L15	L16	L17
Product		pSB1C3-I13453-34-AOX1	pSB1C3-I13453-pSB1C3-BBa_B0032_cbtA	pSB1C3-oHybB-pSB1C3-E0240	pSB1C3-cspD	pSB1C3-accD	pSB1C3-ldhA	pSB1C3-ompT	pSB1C3-RNAG120

Vector	Vector	pSB1C3-I13453	pSB1C3-I13453	pSB1C3-oHybB	pSB1C3	pSB1C3	pSB1C3	pSB1C3	pSB1C3
	Tube	16	16	5	7	7	7	7	7
	ρ	11 ng/µL	11 ng/µL	28 ng/µL	19 ng/µL	19 ng/µL	19 ng/µL	19 ng/µL	19 ng/µL
	Länge	2200 bp	2200 bp	2200 bp	2070 bp	2070 bp	2070 bp	2070 bp	2070 bp
	V	1.41 µL	3.26 µL	0.72 µL	2.54 µL	0.97 µL	0.91 µL	0.94 µL	3.33 µL
Part 1	Part 1	34-AOX1	pSB1C3-BBa_B0032_cbtA	pSB1C3-E0240	cspD	accD	ldhA	ompT	RNAG120
	Tube	6	17	2	13	19	20	21	22
	ρ	3 ng/µL	5 ng/µL	8 ng/µL	10 ng/µL	10 ng/µL	10 ng/µL	10 ng/µL	10 ng/µL
	Länge	1200 bp	394 bp	878 bp	222 bp	915 bp	987 bp	954 bp	120 bp
	V	33.80 µL	12.83 µL	10.00 µL	5.17 µL	8.16 µL	8.27 µL	8.22 µL	3.67 µL
Part 2	Part 2
	Tube
	ρ
	Länge
	V
T4 Buffer		4.00 µL	2.00 µL	2.00 µL	1.00 µL	2.00 µL	2.00 µL	2.00 µL	1.00 µL
T4 Ligase		0.50 µL	0.50 µL	0.50 µL	0.50 µL	0.50 µL	0.50 µL	0.50 µL	0.50 µL
H2O		0.29 µL	1.41 µL	6.79 µL	0.79 µL	8.37 µL	8.32 µL	8.34 µL	1.50 µL
ad		40.00 µL	20.00 µL	20.00 µL	10.00 µL	20.00 µL	20.00 µL	20.00 µL	10.00 µL

The probes were incubated for 1.5h at 37°C.

The products were transformed into DH5α.

150 µl Competent cell aliquots were thawed on ice for 15-30 min.
10 µl DNA were added and mixed
Cells were incubated for 35 min on ice
Heat-shock: 42°C for 45 sec, then put back on ice for 2-5 min
Pre-warm 850 µl LB-Medium
Add pre-warmed LB-Medium into the competent cells. 37°C 900 rpm 1 h
Cetrifuge 4500 rpm 2 min to collect cells.
Put cells on appropriate plates or medium
Incubation at 37°C overnight.

PCR to amplify pSB1C3 and pSB1K3

2 samples of each part were set up.

Mastermix

Substances	1x volume (µl)	5x volume (µl)
DNA-Template	0.1	0.5
F-Primer	0.35	1.75
R-Primer	0.35	1.75
5x Phusion HF-buffer	10	50
dNTPs	1	5
Phusion polymerase	0.5	2.5
ddH₂O	37.7	188.5

PCR-Program (30 cycles)

Temperature (°C)	Time (s)
98	120
98	30
55	30
72	180
72	600

5 µl of each product was applied to a 1% agarose gel. The rest was purified using the “GeneJET Gel Extraction Kit” of Thermo Fisher.

https://static.igem.org/mediawiki/2018/c/cd/T--Hamburg--180930PCR_test.png

PCR of cspD

Mastermix

Substances	1x volume (µl)	5x volume (µl)
DNA-Template	0.1	0.5
F-Primer	0.35	1.75
R-Primer	0.35	1.75
5x Phusion HF-buffer	10	50
dNTPs	1	5
Phusion polymerase	0.5	2.5
ddH₂O	37.7	188.5

PCR-Program (30 cycles)

Temperature (°C)	Time (s)
98	120
98	30
55	30
72	180
72	600

https://static.igem.org/mediawiki/2018/1/1c/T--Hamburg--180930_cPCR_Pcr_test.png

Colony PCR of pSB1C3-BBa_I732018

The transformed part set up on September 29 was used.

Mastermix

Substance	1x (volume in µl)	5x (volume in µl)
10x DreamTaq Buffer	2	10
dNTPs 10mM	0.4	2
MgCl₂ 50mM	1.2	6
Fw-Primer VF2	0.6	3
Rv-Primer VR	0.6	3
Taq-Polymerase	0.1	0.5
ddH₂O ad 20µl	15.1	75.5

Colonies were picked with pipette-tips and transferred into 1 ml LB-Medium tubes after they were put in 20 µl aliquots of the mastermix of the following PCR reaction

PCR-Program (30 cycles)

Temperature (°C)	Time (s)
95	180
95	30
55	30
72	150
72	180

The products were applied to a 1% agarose gel.

https://static.igem.org/mediawiki/2018/1/1c/T--Hamburg--180930_cPCR_Pcr_test.png

PCR for the production of pSB1C3 and pSB1K3

The PCR reactions were prepared as follows:

	1x
5x Phusion HF-Buffer	10 µL
dNTPs	1 µL
SB-Prep-3P1	0.35 µL
SB-Prep-2Ea	0.35 µL
Phusion Polymerase	0.5 µL
H2O (ad 50)	37.7 µL
Template (je 2x pSB1K3 und psB1C3)	0.1 µL

The PCR programme was as follows:

Temperatur	Zeit
98 °C	2:00
98 °C	0:30
55 °C	0:30
72 °C	3:00
go to step to (30x)
72 °C	10:00
4 °C	∞

Restriction and ligation 1

The restriction reactions were prepared as follows:

Tube

1

2

3

4

5

6

7

8

9

10

11

12

13

14

15

16

17

18

19

20

21

22

Part

pSB1C3_RNAG120_MlcRE

pSB1C3-E0240

BBa_J45503

pSB1C3-oHybB

34-AOX1

pSB1C3

mraz

pSB1C3-BBa_K206000

31sulA

32sulA

34sulA

cspD

pSB1C3-BBa_B0032

pSB1C3

pSB1C3-I13453

pSB1C3-BBa_B0032_cbtA

pSB1C3-BBa_I732018

accD

ldhA

ompT

RNAG120

Lot#

180925NaF01

180925NaF02

180817LD10

180625NiF12

180921OM03

180920LP01

180914LP01

180622NB08

neue Miniprep

180905AW03

180622NB04

neue Miniprep

180924AW1

180925NaF04

180926NaF03

180921BK09

180622NB01

180827LP01

180909BK09

ρ

140 ng/µL

144.673

200 ng/µL

45 ng/µL

186 ng/µL

575 ng/µL

89 ng/µL

44.175

50 ng/µL

28 ng/µL

40 ng/µL

43 ng/µL

50 ng/µL

81 ng/µL

95 ng/µL

126 ng/µL

93.011

213 ng/µL

57 ng/µL

68 ng/µL

77 ng/µL

Use

Vector

vector

single insert

vector

single insert

vector

single insert

vector

single insert

vector

single insert

target m

1000 ng

100 ng

1000 ng

100 ng

1000 ng

100 ng

1000 ng

100 ng

1000 ng

100 ng

Volumes

DNA

7.15 µL

6.91 µL

5.00 µL

2.24 µL

5.38 µL

0.17 µL

11.24 µL

2.26 µL

20.20 µL

3.57 µL

2.50 µL

2.33 µL

2.00 µL

12.42 µL

10.57 µL

7.93 µL

10.75 µL

0.47 µL

1.77 µL

1.47 µL

1.30 µL

NEBuffer 2.1

1.00 µL

4.00 µL

2.00 µL

1.00 µL

3.00 µL

1.00 µL

2.00 µL

1.00 µL

EcoRI-HF

0.50 µL

XbaI

0.50 µL

SpeI

0.50 µL

PstI

0.50 µL

FastAP

0.50 µL

H2O

0.35 µL

1.09 µL

2.50 µL

5.76 µL

2.12 µL

34.83 µL

5.26 µL

5.74 µL

5.30 µL

4.43 µL

5.50 µL

5.67 µL

6.00 µL

4.08 µL

5.93 µL

8.57 µL

6.25 µL

8.00 µL

7.53 µL

6.23 µL

6.53 µL

6.70 µL

ad

10.00 µL

40.00 µL

20.00 µL

10.00 µL

30.00 µL

10.00 µL

20.00 µL

10.00 µL

Länge Produkt

2697 bp

878 bp

2200 bp

1200 bp

2070

222

2070

2200

394

252

915

987

954

120

ρ final

8 ng/µL

6 ng/µL

10 ng/µL

28 ng/µL

3 ng/µL

19 ng/µL

10 ng/µL

6 ng/µL

10 ng/µL

8 ng/µL

50 ng/µL

11 ng/µL

5 ng/µL

0 ng/µL

10 ng/µL

The ligations were prepared as follows:

Tube

L1

L2

L3

L4

L5

L6

L7

L8

L9

L10

L11

L12

L13

L14

L15

L16

L17

Product

pSB1C3_RNAG120_MlcRE-pSB1C3-E0240

pSB1C3-E0240-BBa_J45503

pSB1C3-oHybB-34-AOX1

pSB1C3-mraz

pSB1C3-BBa_K206000-32sulA

pSB1C3-BBa_K206000-34sulA

pSB1C3-BBa_B0032-cspD

pSB1C3-34-AOX1

pSB1C3-I13453-34-AOX1

pSB1C3-I13453-pSB1C3-BBa_B0032_cbtA

pSB1C3-oHybB-pSB1C3-E0240

pSB1C3_RNAG120_MlcRE-pSB1C3-BBa_I732018

pSB1C3-cspD

pSB1C3-accD

pSB1C3-ldhA

pSB1C3-ompT

pSB1C3-RNAG120

Vector

Vector

pSB1C3_RNAG120_MlcRE

pSB1C3-E0240

pSB1C3-oHybB

pSB1C3

pSB1C3-BBa_K206000

pSB1C3-BBa_B0032

pSB1C3

pSB1C3-I13453

pSB1C3-oHybB

pSB1C3_RNAG120_MlcRE

pSB1C3

Tube

1

3

5

7

9

14

15

16

5

1

7

ρ

8 ng/µL

10 ng/µL

28 ng/µL

19 ng/µL

6 ng/µL

8 ng/µL

50 ng/µL

11 ng/µL

28 ng/µL

8 ng/µL

19 ng/µL

Länge

2697 bp

2200 bp

2070 bp

2200 bp

2697 bp

2070 bp

V

2.97 µL

0.55 µL

0.61 µL

4.25 µL

0.00 µL

0.29 µL

1.41 µL

3.26 µL

0.72 µL

6.54 µL

2.54 µL

0.97 µL

0.91 µL

0.94 µL

3.33 µL

Part 1

Part 1

pSB1C3-E0240

BBa_J45503

34-AOX1

mraz

32sulA

34sulA

cspD

34-AOX1

pSB1C3-BBa_B0032_cbtA

pSB1C3-E0240

pSB1C3-BBa_I732018

cspD

accD

ldhA

ompT

RNAG120

Tube

2

4

6

8

11

12

13

6

17

2

18

13

19

20

21

22

ρ

6 ng/µL

10 ng/µL

3 ng/µL

10 ng/µL

3 ng/µL

5 ng/µL

6 ng/µL

10 ng/µL

Länge

878 bp

1200 bp

222 bp

1200 bp

394 bp

878 bp

252 bp

222 bp

915 bp

987 bp

954 bp

120 bp

V

12.75 µL

33.80 µL

10.00 µL

34.12 µL

33.80 µL

12.83 µL

12.97 µL

5.17 µL

8.16 µL

8.27 µL

8.22 µL

3.67 µL

Part 2

Part 2

Tube

ρ

Länge

V

T4 Buffer

2.00 µL

1.00 µL

4.00 µL

2.00 µL

1.00 µL

2.00 µL

4.00 µL

2.00 µL

1.00 µL

2.00 µL

1.00 µL

T4 Ligase

0.50 µL

H2O

1.78 µL

8.50 µL

1.14 µL

6.89 µL

5.63 µL

7.50 µL

1.09 µL

0.29 µL

1.41 µL

3.82 µL

1.96 µL

0.79 µL

8.37 µL

8.32 µL

8.34 µL

1.50 µL

ad

20.00 µL

10.00 µL

40.00 µL

20.00 µL

10.00 µL

20.00 µL

40.00 µL

20.00 µL

10.00 µL

20.00 µL

10.00 µL

The Gel extration of pSB1C3-RNAG120-MlcRE (~2100 bp) & E0240 (Bande bei ~900 bp) was performed according to the kit’s protocol. Nanodrop measurement showed results as follows:

181001DDW01 pSB1C3-RNAG120-MlcRE : 7.903 ng/µL

181001DDW02 E0240 : 6.166 ng/µL

The concentrations measured after the PCR purification were as follows:

pSB1C3: 25.1 ng/µL

pSB1K3: 41.411 ng/µL

L1 was transformed and incubated at 37°C overnight.

cPCR for golden gate assemblies

The reactions and the results of the GGA are summarised in the following table:

Part	fw-Primer	rv-Primer	Template	Tube	concentration (ng/µL)
accC	BsaI-accC-f	BsaI-accC-r	180925BK01	1	multiple bands
accD	BsaI-accD-f	BsaI-accD-r	180925BK02	2	did not work
ADH2	BsaI-ADH2-f	BsaI-ADH2-r	180925BK03	3	multiple bands
kivD	BsaI-kivD-f	BsaI-kivD-r	180925BK04	4	did not work
pBAD	dapAP-BsaI-f	BsaI-pBAD-r	180925BK05	5
HlyB	BsaI-HlyB-f	BsaI-HlyB-r	180925BK06	6	did not work
RnaG120 + Terminator	dapAP-BsaI-f	RnaG120-r	180925NaF01	7

The PCR programme was performed as follows:

Temperature	Time	Steps
98	0:20	1
98	0:10	30
60	0:20	30
72	2:00	30
72	5:00	1

PCR Purification of Tube 5 (181001BK01) and Tube 7 (181001BK02).

cPCR of the transformation from the 30.09.2018

The cPCR reactions were prepared as follows:

	1x	65x
10x DreamTaq Buffer	2 µL	130 µL
dNTPs	0.4 µL	26 µL
VF2	0.6 µL	39 µL
VR	0.6 µL	39 µL
MgCl2 50 mM	1.2 µL	78 µL
Dream Taq	0.5 µL	32.5 µL
H2O (ad 20)	15.1 µL	981.5 µL
Template (von Platten)	0.1 µL

The cPCR programme was performed as follows:

Temperatur	Zeit
98 °C	3:00
98 °C	0:30
55 °C	0:30
72 °C	1:30
go to step to (30x)
72 °C	5:00
4 °C	∞

The marked clones were used to prepare an overnight culture.

181001 – 7

Overnight culture of pBAD-AOX1 (180908NF01) for AOX1 characterisation

The cryostock 180908NF01 was used to set up an ONC.

181002 – 1

Characterisation of AOX1

We have a room temperature of 21.5 °C. The incubator shows a temperature of 25.6 °C. Cultures of 100ml were set up at an OD of 0.1. Induced at 120 min (and respective OD at 120 minutes) with 1 mL of 0.1 mol/L Arabinose. After measurement at 150 min, temperature and OD were measured in 1 h intervalls. After measurement of 570 min, 2 mL of culture was transferred into New medium (100 mL) and Arabinose (1 mL, 0.1 mM) was added to the induced cultures. Cultures were incubated overnight at 16*C to control for late induction of the promoter.

3 x 100 mL pBAD-AOX1 (induced)

1.

Time	OD	Temperature (°C)
0	0.1	15.5
30	0.13	24
60	0.138	25
90	0.184	26
120	0.234	26
150	0.274	25.5
210	0.568	27.5
270	0.968	28
330	1.960	28
390	2.260	28
450	2.76	28.5
510	3.26 (contaminated)	28.5
570	2.66	28.5

2.

Time	OD	Temperature (°C)
0	0.1	15.5
30	0.13	24
60	0.14	25
90	0.164	26
120	0.202	26
150	0.244	25.5
210	0.396	27
270	0.786	28
330	1.96	28
390	2.26	28
450	2.80	28.5
510	3.52 (contaminated)	28.5
570	3.30	28

3.

Time	OD	Temperature (°C)
0	0.1	15.5
30	0.14	23.5
60	0.156	26
90	0.196	26
120	0.276	26
150	0.314	25
210	0.660	27.5
270	0.920	28
330	2.06	28
390	2.36	28
450	3.40	28
510	3.28 (contaminated)	28
570	3.30	28

1 x 100 mL pBAD-AOX1 (not induced)

Time	OD	Temperature (°C)
0	0.1	15
30	0.11	24
60	0.122	25
90	0.178	26
120	0.190	26
150	0.266	25
210	0.442	27
270	0.956	27
330	2.18	27
390	2.98	27.5
450	3.38	28
510	4.36 (contaminated)	28
570	3.24	27.5

1 x 100 mL DH5a

Time	OD	Temperature (°C)
0	0.1
30	0.11	23.5
60	0.116	26
90	0.152	25.5
120	0.176	26
150	0.198	24.5
210	0.385	26.5
270	0.764	27
330	2.0	27
390	2.60	27
450	3.38	27.5
510	3.97 (contaminated)	27.5
570	3.90	27.5

181002 – 2

PCR of pSB1C3 and missing GGA parts

The plan for the PCR was as follows:

Part	fw-Primer	rv-Primer	Template	Tube	result
BsaI-ADH2	BsaI-ADH2-f	BsaI-ADH2-r	180726LP03	1	wrong length
BsaI-kivD	BsaI-kivD-f	BsaI-kivD-r	180726LP01	2	no bands
BsaI-HlyB	BsaI-HlyB-f	BsaI-HlyB-r	K554013	3	multiple bands
pSB1C3	SB-Prep-3p1	SB-Prep-2ea	pSB1C3 aus Kit	4	no bands

For HlyB gel extraction was performed.

The PCR programme was as follows:

Temperature	Time	Steps
98	0:20	1
98	0:10	30
60	0:20	30
72	2:30 (pSB1C3 2:00)	30
72	5:00	1

The PCR was prepared as follows:

Volume (µL)	Chemical
20	H2O
2.5	rv-Primer
2.5	fw-Primer
25	Q5 2X Master Mix
0.25 (pSB1C3 0.5)	Template

181002 – 3

Minipreps

Minipreps were performed according to the kit’s protocol. The nanodrop measured the concentration as follows:

LOT number	part	concentration [ng/µL]
181002NB06	pSB1C3-cspD	76.138
181002NB07	pSB1C3-oHybB-BBa_E0240	150.571
181002NB08	pSB1C3-BBa_I13453-BBa_0032-cbtA	157.52
181002NB09	pSB1C3-ldhA	65.203
181002NB10	pSB1C3-ompt	106.305

181002 – 4

Preparation for Sequencing

overall low visibility in the control restriction and light bands

180726LP04 → psB1C3_accC #10 → too short

180920OM01 → pSB1C3_OmpA-Aox1 #17 → smear

180914AW02 → pSB1C3-MlcRE → no band

181002NB06 → pSB1C3-cspD → no band

181002NB07 → pSB1C3-oHybB-E0240 → no band

181002NB08 → pSB1C3-BBa_I13453-BBa_0032-cbtA → light band (little high but could be right (vf2 vr)

181002NB09 → pSB1C3-ldhA → light right band (vf2 vr)

181002NB010 → pSB1C3-ompT → no band

180912LP02 → pGGA-NotMlcRE-HlyB-HlyD-TolC-OmpT → no band

180920OM02 → pGGA-NotMlcRE-HlyB-HlyD-TolC-OmpT (#16) → multiple bands, one right band → retrafo

compound	a-h	i and j
DNA	5 µL	5 µL
EcoRI	0.5 µL
BsaI		0.5 µL
FastAP	0.5 µL	0.5 µL
Buffer 2.1	1 µL
Buffer CutSmart		1 µL
H2O	3 µL	3 µL

181002 – 5

Restriction, ligation and transformation of multiple parts

The Restriction was prepared as follows:

Tube

1

2

3

4

5

6

7

8

9

10

11

12

13

14

15

16

17

18

19

20

21

22

23

24

25

26

27

Part

pSB1C3_RNAG120_MlcRE

pSB1C3-E0240

BBa_J45503

pSB1C3-oHybB

34-AOX1

pSB1C3

mraz

pSB1C3-BBa_K206000

31sulA

32sulA

34sulA

cspD

pSB1C3-BBa_B0032

pSB1C3

pSB1C3-I13453

pSB1C3-BBa_B0032_cbtA

pSB1C3-BBa_I732018

accD

ldhA

ompT

RNAG120

pSB1K3

pSB1A3-BBa_B0034

pGGA-NotMlcRE-HlyB-HlyD-TolC-OmpT

pGGA-pBAD-BjalT-OmpT-FLAG-HlyA

Lot#

180925NaF01

180925NaF02

180817LD10

180625NiF12

181001NB05

180920LP01

180914LP01

180622NB08

neue Miniprep

180905AW03

180622NB04

neue Miniprep

180924AW1

180925NaF04

180926NaF03

181001NB03

180921BK09

180622NB01

180827LP01

180909BK09

181001NB07

180514NB02

180912LP02

180920OM02

180803NB02

ρ

140 ng/µL

144.673

200 ng/µL

45 ng/µL

107 ng/µL

575 ng/µL

89 ng/µL

44.175

50 ng/µL

28 ng/µL

40 ng/µL

43 ng/µL

50 ng/µL

81 ng/µL

95 ng/µL

126 ng/µL

93.011

162 ng/µL

213 ng/µL

57 ng/µL

68 ng/µL

77 ng/µL

41.411

65

129.25

800 ng/µL

235.7

Use

Vector

vector

single insert

vector

single insert

vector

single insert

vector

single insert

vector

single insert

Vector

target m

1000 ng

100 ng

1000 ng

100 ng

1000 ng

100 ng

1000 ng

100 ng

1000 ng

100 ng

1000 ng

Volumes

DNA

7.15 µL

6.91 µL

5.00 µL

2.24 µL

9.34 µL

0.17 µL

11.24 µL

2.26 µL

20.20 µL

3.57 µL

2.50 µL

2.33 µL

2.00 µL

12.42 µL

10.57 µL

7.93 µL

10.75 µL

6.17 µL

0.47 µL

1.77 µL

1.47 µL

1.30 µL

24.15 µL

15.38 µL

7.74 µL

2.50 µL

8.49 µL

NEBuffer 2.1

1.00 µL

2.00 µL

4.00 µL

2.00 µL

1.00 µL

3.00 µL

1.00 µL

2.00 µL

1.00 µL

3.00 µL

2.00 µL

1.00 µL

2.00 µL

EcoRI-HF

0.50 µL

0.60 µL

0.50 µL

XbaI

0.50 µL

SpeI

0.50 µL

PstI

0.50 µL

0.60 µL

0.50 µL

FastAP

0.50 µL

H2O

0.85 µL

0.59 µL

2.50 µL

5.76 µL

7.66 µL

34.83 µL

5.26 µL

5.74 µL

5.30 µL

4.43 µL

5.50 µL

5.67 µL

6.00 µL

4.08 µL

5.93 µL

8.57 µL

6.25 µL

1.83 µL

7.53 µL

6.23 µL

6.53 µL

6.70 µL

1.15 µL

1.12 µL

9.26 µL

5.50 µL

8.51 µL

ad

10.00 µL

20.00 µL

40.00 µL

20.00 µL

10.00 µL

30.00 µL

10.00 µL

20.00 µL

10.00 µL

30.00 µL

20.00 µL

10.00 µL

20.00 µL

Länge Produkt

2697 bp

878 bp

2200 bp

1200 bp

2070

222

2070

2200

394

252

915

987

954

120

2070

6500

602

ρ final

7 ng/µL

6 ng/µL

Gelex

3 ng/µL

50 ng/µL

10 ng/µL

6 ng/µL

10 ng/µL

8 ng/µL

50 ng/µL

7 ng/µL

50 ng/µL

100 ng/µL

10 ng/µL

33 ng/µL

Gelex

The ligation was prepared as follows:

Tube

L1

L2

L3

L4

L5

L6

L7

L8

L9

L10

L11

L12

L13

L14

L15

L16

L17

L18

L19

L20

L21

L22

L23

L24

L25

L26

Product

pSB1C3_RNAG120_MlcRE-E0240

pSB1C3-E0240-BBa_J45503

pSB1C3-oHybB-34-AOX1

pSB1C3-mraz

pSB1C3-BBa_K206000-32sulA

pSB1C3-BBa_K206000-34sulA

pSB1C3-BBa_B0032-cspD

pSB1C3-34-AOX1

pSB1C3-I13453-34-AOX1

pSB1C3-I13453-pSB1C3-BBa_B0032_cbtA

pSB1C3-oHybB-E0240

pSB1C3_RNAG120_MlcRE-pSB1C3-BBa_I732018

pSB1C3-cspD

pSB1C3-accD

pSB1C3-ldhA

pSB1C3-ompT

pSB1C3-RNAG120

pSB1C3-NotMlcRE-HlyB-HlyD-TolC-OmpT

pSB1C3-pBAD-BjalT-OmpT-FLAG-HlyA

pSB1K3-NotMlcRE-HlyB-HlyD-TolC-OmpT

pSB1K3-pBAD-BjalT-OmpT-FLAG-HlyA

pSB1A3-NotMlcRE-HlyB-HlyD-TolC-OmpT

pSB1A3-pBAD-BjalT-OmpT-FLAG-HlyA

Vector

Vector

pSB1C3_RNAG120_MlcRE

pSB1C3-E0240

pSB1C3-oHybB

pSB1C3

pSB1C3-BBa_K206000

pSB1C3-BBa_B0032

pSB1C3

pSB1C3-I13453

pSB1C3-oHybB

pSB1C3_RNAG120_MlcRE

pSB1C3

pSB1K3

pSB1A3-BBa_B0034

Tube

1

3

5

7

9

14

15

16

5

1

7

23

24

ρ

7 ng/µL

Gelex

50 ng/µL

6 ng/µL

8 ng/µL

50 ng/µL

7 ng/µL

Gelex

7 ng/µL

50 ng/µL

33 ng/µL

Gelex

Länge

2697 bp

2200 bp

2070 bp

2200 bp

2697 bp

2070 bp

V

3.15 µL

0.61 µL

4.25 µL

0.00 µL

0.29 µL

2.21 µL

5.12 µL

6.93 µL

0.97 µL

0.37 µL

0.35 µL

0.36 µL

1.27 µL

0.06 µL

0.12 µL

1.02 µL

0.19 µL

1.54 µL

Part 1

Part 1

pSB1C3-E0240

BBa_J45503

34-AOX1

mraz

32sulA

34sulA

cspD

34-AOX1

pSB1C3-BBa_B0032_cbtA

pSB1C3-E0240

pSB1C3-BBa_I732018

cspD

accD

ldhA

ompT

RNAG120

pGGA-NotMlcRE-HlyB-HlyD-TolC-OmpT

pGGA-pBAD-BjalT-OmpT-FLAG-HlyA

pGGA-NotMlcRE-HlyB-HlyD-TolC-OmpT

pGGA-pBAD-BjalT-OmpT-FLAG-HlyA

pGGA-NotMlcRE-HlyB-HlyD-TolC-OmpT

pGGA-pBAD-BjalT-OmpT-FLAG-HlyA

Tube

2

4

6

8

11

12

13

6

17

2

18

13

19

20

21

22

25

26

27

25

26

27

25

26

27

ρ

6 ng/µL

Gelex

3 ng/µL

10 ng/µL

3 ng/µL

50 ng/µL

6 ng/µL

100 ng/µL

10 ng/µL

Gelex

Länge

878 bp

1200 bp

222 bp

1200 bp

394 bp

878 bp

252 bp

222 bp

915 bp

987 bp

954 bp

120 bp

6500 bp

602 bp

6500 bp

602 bp

6500 bp

602 bp

V

6.00 µL

33.80 µL

10.00 µL

34.12 µL

33.80 µL

1.28 µL

12.97 µL

0.48 µL

5.17 µL

8.16 µL

8.27 µL

8.22 µL

3.67 µL

Part 2

Part 2

Tube

ρ

Länge

V

T4 Buffer

2.00 µL

1.00 µL

4.00 µL

2.00 µL

1.00 µL

2.00 µL

4.00 µL

5.00 µL

1.00 µL

2.00 µL

1.00 µL

2.00 µL

1.00 µL

T4 Ligase

0.50 µL

H2O

8.35 µL

8.50 µL

1.70 µL

6.89 µL

5.63 µL

7.50 µL

1.09 µL

8.48 µL

2.10 µL

4.53 µL

1.09 µL

2.36 µL

-0.02 µL

8.89 µL

-0.07 µL

3.56 µL

8.44 µL

8.38 µL

7.48 µL

8.31 µL

6.96 µL

8.50 µL

ad

20.00 µL

10.00 µL

40.00 µL

20.00 µL

10.00 µL

20.00 µL

40.00 µL

50.00 µL

10.00 µL

20.00 µL

10.00 µL

20.00 µL

10.00 µL

Only L1 was performed.

Overnight cultures

a) Overnight cultures were prepared from the minipreps of this morning, because gDNA was visible in the test digest

LOT Nummer Plasmid	Insert	LOT Nummer Cryostock
180726LP04	psB1C3_accC #10	180727DK04
180920OM01	pSB1C3_OmpA-Aox1 #17	No cryostock available
180914AW02	pSB1C3-MlcRE	180908NF03
181002NB06	pSB1C3-cspD	181002NB01
181002NB07	pSB1C3-oHybB-E0240	181002NB02
181002NB08	pSB1C3-BBa_I13453-BBa_0032-cbtA	181002NB03
181002NB09	pSB1C3-ldhA	181002NB04
181002NB010	pSB1C3-ompT	181002NB05
180912LP02	pGGA-NotMlcRE-HlyB-HlyD-TolC-OmpT	180912LP01
180920OM02	pGGA-NotMlcRE-HlyB-HlyD-TolC-OmpT (#16)	180917AK02

c) Overnight culture of pSB1C3-BBa_K206000-BBa_0034-ldhA for production of cell lysates for the characterisation on Wednesday → 180518BK11
d) Check, if there are still enough sample for restriction for tomorrow, otherwise new overnight cultures need to be prepared. Result: everything is empty, so new overnight cultures were prepared from the following lot numbers:

180912LP01

180917AK02

180803NB01 → pGGA-pBAD-BjalT-OmpT-FLAG-HlyA

181003 – 1

Characterisation of AOX1

Reactions from yesterday were cultured overnight at 16 °C and shook.

3 x 100 mL pBAD-AOX1 (induced)

1.

Time [am]	OD	Temperature (°C)
9:30		17.5
10:30		18
11:30	1.88	18

2.

Time [am]	OD	Temperature (°C)
9:30		18.5
10:30		18
11:30	1.60	18

3.

Time [am]	OD	Temperature (°C)
9:30		18
10:30		18
11:30	2.10	18.5

1x 100ml pBAD-AOX1 not induced

Time [am]	OD	Temperature (°C)
9:30		18
10:30		18
11:30	2.66	18

1x 100ml DH5a

Time [am]	OD	Temperature (°C)
9:30		18
10:30		18.2
11:30	2.74	19

The experiment was cancelled after 11:30 h, because there was no observable change anymore.

181003 – 2

Miniprep purification of the overnight cultures of yesterday

The products of the minipreps got new lot numbers afterwards:

181003NB01	03.10.2018	pGGA-NotMlcRE-HlyB-HlyD-TolC-OmpT (#16)
181003NB02	03.10.2018	pSB1C3-BBa_I13453-BBa_0032-cbtA
181003NB03	03.10.2018	pSB1C3-MlcRE
181003NB04	03.10.2018	pGGA-pBAD-BjalT-OmpT-FLAG-HlyA
181003NB05	03.10.2018	psB1C3_accC #10
181003NB06	03.10.2018	pSB1C3-oHybB-BBa_E0240
181003NB07	03.10.2018	pSB1C3-cspD
181003NB08	03.10.2018	pGGA-NotMlcRE-HlyB-HlyD-TolC-OmpT
181003NB09	03.10.2018	pSB1C3-ldhA
181003NB10	03.10.2018	pSB1C3-ompt

181003 – 3

Restriction, ligation, transformation

The restriction was prepared as follows:

Tube

1

2

3

4

5

6

7

8

9

10

11

12

13

14

15

16

17

18

19

20

21

22

23

24

25

26

27

28

29

30

Part

pSB1C3_RNAG120_MlcRE

pSB1C3-E0240

BBa_J45503

pSB1C3-oHybB

34-AOX1

pSB1C3

mraz

pSB1C3-BBa_K206000

31sulA

32sulA

34sulA

cspD

pSB1C3-BBa_B0032

pSB1C3

pSB1C3-I13453

pSB1C3-BBa_B0032_cbtA

pSB1C3-BBa_I732018

accD

ldhA

ompT

RNAG120

pSB1K3

pSB1A3-BBa_B0034

pGGA-NotMlcRE-HlyB-HlyD-TolC-OmpT

pGGA-pBAD-BjalT-OmpT-FLAG-HlyA

pSB1K3

pSB1C3

pSB1A3-BBa_B0034

Lot#

180925NaF01

180925NaF02

180817LD10

180625NiF12

181001NB05

180920LP01

backbone

180622NB08

neue Miniprep

180905AW03

180622NB04

neue Miniprep

180924AW1

180925NaF04

180926NaF03

181001NB03

180921BK09

180622NB01

180827LP01

180909BK09

backbone

180514NB02

180912LP02

180920OM02

181003NB04

backbone

180514NB02

ρ

140 ng/µL

144.673

200 ng/µL

45 ng/µL

107 ng/µL

575 ng/µL

25 ng/µL

44.175

50 ng/µL

28 ng/µL

40 ng/µL

43 ng/µL

50 ng/µL

81 ng/µL

95 ng/µL

126 ng/µL

93.011

162 ng/µL

213 ng/µL

57 ng/µL

68 ng/µL

77 ng/µL

25 ng/µL

65

129 ng/µL

800 ng/µL

267 ng/µL

25 ng/µL

65

Use

Vector

vector

single insert

vector

single insert

vector

single insert

vector

single insert

vector

single insert

vector

target m

1000 ng

100 ng

1000 ng

100 ng

500 ng

100 ng

1000 ng

100 ng

1000 ng

100 ng

200 ng

1000 ng

2000 ng

100 ng

200 ng

Volumes

DNA

7.15 µL

6.91 µL

5.00 µL

2.24 µL

9.34 µL

0.17 µL

20.00 µL

2.26 µL

20.20 µL

3.57 µL

2.50 µL

2.33 µL

2.00 µL

12.42 µL

10.57 µL

7.93 µL

10.75 µL

12.34 µL

0.47 µL

1.77 µL

1.47 µL

1.30 µL

8.00 µL

30.77 µL

15.47 µL

2.50 µL

20.00 µL

8.00 µL

NEBuffer 2.1

1.00 µL

2.00 µL

4.00 µL

3.00 µL

1.00 µL

3.00 µL

1.00 µL

2.00 µL

1.00 µL

2.00 µL

4.00 µL

2.00 µL

1.00 µL

3.00 µL

2.00 µL

EcoRI-HF

0.50 µL

XbaI

0.50 µL

SpeI

0.50 µL

PstI

0.50 µL

FastAP

0.50 µL

H2O

0.85 µL

0.59 µL

2.50 µL

5.76 µL

7.66 µL

34.83 µL

5.50 µL

5.74 µL

5.30 µL

4.43 µL

5.50 µL

5.67 µL

6.00 µL

4.08 µL

5.93 µL

8.57 µL

6.25 µL

4.66 µL

7.53 µL

6.23 µL

6.53 µL

6.70 µL

8.50 µL

3.73 µL

1.53 µL

5.50 µL

6.00 µL

8.50 µL

ad

10.00 µL

20.00 µL

40.00 µL

30.00 µL

10.00 µL

30.00 µL

10.00 µL

20.00 µL

10.00 µL

20.00 µL

40.00 µL

20.00 µL

10.00 µL

30.00 µL

20.00 µL

Länge Produkt

2697 bp

878 bp

2200 bp

1200 bp

2070

222

2070

2200

394

252

915

987

954

120

2070

6500

602

2070

ρ final

Gelex

17 ng/µL

Gelex

5 ng/µL

Gelex

30 ng/µL

10 ng/µL

33 ng/µL

10 ng/µL

8 ng/µL

50 ng/µL

8 ng/µL

7 ng/µL

10 ng/µL

19 ng/µL

16 ng/µL

Gelex

11 ng/µL

16 ng/µL

14 ng/µL

26 ng/µL

The restriction was performed at 37 °C for 1h followed by 80 °C for 20 min.

Afterwards, the samples were put on a 1 % agarose gel for gel electrophoresis.

pGGA-pBAD-BjalT-OmpT-FLAG-HlyA was cut three times, because the double digest results in two fragments of the same size, of which one is the desired one and the second part of the vector (third enzyme NcoI cuts only in the vector fragment)

a double restriction reaction is necessary, because NcoI is available only from Thermo Fisher

Framed bands were cut out and used for ligation.

Samples 180518BK15, 181001NB02 were empty and were re-produced with a new overnight culture.

Second digest of pGGA-pBAD-BjalT-OmpT-FLAG-HlyA, each upper band was cut out for ligation

The ligation was prepared as follows:

Tube

L1

L2

L3

L4

L5

L6

L7

L8

L9

L10

L11

L12

L13

L14

L15

L16

L17

L18

L19

L20

L21

L22

L23

L24

L25

L26

Product

pSB1C3_RNAG120_MlcRE-pSB1C3-E0240

pSB1C3-E0240-BBa_J45503

pSB1C3-oHybB-34-AOX1

pSB1C3-mraz

pSB1C3-BBa_K206000-32sulA

pSB1C3-BBa_K206000-34sulA

pSB1C3-BBa_B0032-cspD

pSB1C3-34-AOX1

pSB1C3-I13453-34-AOX1

pSB1C3-I13453-pSB1C3-BBa_B0032_cbtA

pSB1C3-oHybB-pSB1C3-E0240

pSB1C3_RNAG120_MlcRE-pSB1C3-BBa_I732018

pSB1C3-cspD

pSB1C3-accD

pSB1C3-ldhA

pSB1C3-ompT

pSB1C3-RNAG120

pSB1C3-NotMlcRE-HlyB-HlyD-TolC-OmpT

pSB1C3-pBAD-BjalT-OmpT-FLAG-HlyA

pSB1K3-NotMlcRE-HlyB-HlyD-TolC-OmpT

pSB1K3-pBAD-BjalT-OmpT-FLAG-HlyA

pSB1A3-NotMlcRE-HlyB-HlyD-TolC-OmpT

pSB1A3-pBAD-BjalT-OmpT-FLAG-HlyA

Vector

Vector

pSB1C3_RNAG120_MlcRE

pSB1C3-E0240

pSB1C3-oHybB

pSB1C3

pSB1C3-BBa_K206000

pSB1C3-BBa_B0032

pSB1C3

pSB1C3-I13453

pSB1C3-oHybB

pSB1C3_RNAG120_MlcRE

pSB1C3

pSB1K3

pSB1A3-BBa_B0034

Tube

1

3

5

7

9

14

15

16

5

1

7

29

23

28

24

30

ρ

Gelex

5 ng/µL

30 ng/µL

33 ng/µL

8 ng/µL

50 ng/µL

8 ng/µL

5 ng/µL

Gelex

30 ng/µL

14 ng/µL

19 ng/µL

16 ng/µL

26 ng/µL

Länge

2697 bp

2200 bp

2070 bp

2200 bp

2697 bp

2070 bp

V

2.90 µL

0.61 µL

4.25 µL

0.00 µL

0.29 µL

1.92 µL

4.45 µL

3.75 µL

1.62 µL

0.62 µL

0.58 µL

0.60 µL

2.13 µL

0.10 µL

1.83 µL

0.16 µL

1.60 µL

0.19 µL

0.98 µL

Part 1

Part 1

pSB1C3-E0240

BBa_J45503

34-AOX1

mraz

32sulA

34sulA

cspD

34-AOX1

pSB1C3-BBa_B0032_cbtA

pSB1C3-E0240

pSB1C3-BBa_I732018

cspD

accD

ldhA

ompT

RNAG120

pGGA-NotMlcRE-HlyB-HlyD-TolC-OmpT

pGGA-pBAD-BjalT-OmpT-FLAG-HlyA

pGGA-NotMlcRE-HlyB-HlyD-TolC-OmpT

pGGA-pBAD-BjalT-OmpT-FLAG-HlyA

pGGA-NotMlcRE-HlyB-HlyD-TolC-OmpT

pGGA-pBAD-BjalT-OmpT-FLAG-HlyA

Tube

2

4

6

8

11

12

13

6

17

2

18

13

19

20

21

22

25

26

27

25

26

27

25

26

27

ρ

17 ng/µL

Gelex

10 ng/µL

Gelex

8 ng/µL

17 ng/µL

7 ng/µL

10 ng/µL

Gelex

11 ng/µL

Gelex

11 ng/µL

Gelex

11 ng/µL

Länge

878 bp

1200 bp

222 bp

1200 bp

394 bp

878 bp

252 bp

222 bp

915 bp

987 bp

954 bp

120 bp

6500 bp

602 bp

6500 bp

602 bp

6500 bp

602 bp

V

4.54 µL

10.00 µL

8.54 µL

4.75 µL

7.20 µL

5.17 µL

8.16 µL

8.27 µL

8.22 µL

3.67 µL

6.88 µL

Part 2

Part 2

Tube

ρ

Länge

V

T4 Buffer

1.00 µL

2.00 µL

1.00 µL

2.00 µL

1.00 µL

2.00 µL

1.00 µL

2.00 µL

1.00 µL

2.00 µL

1.00 µL

T4 Ligase

0.50 µL

H2O

3.96 µL

8.50 µL

5.60 µL

6.89 µL

5.63 µL

7.50 µL

8.21 µL

6.58 µL

4.51 µL

0.00 µL

1.30 µL

1.70 µL

8.72 µL

8.65 µL

8.68 µL

2.70 µL

8.40 µL

8.79 µL

8.34 µL

0.02 µL

8.31 µL

0.64 µL

ad

10.00 µL

20.00 µL

10.00 µL

20.00 µL

10.00 µL

20.00 µL

10.00 µL

20.00 µL

10.00 µL

20.00 µL

10.00 µL

181003 – 4

cPCR of the two plates pSB1C3-NOT-MlcRE-E0240

One plate was from yesterday, one from the 01.10.

The master mix was prepared as follows:

	1x	22x
10x Dream Taq Buffer	2 µL	44 µL x
dNTPs 10 mM	0.4 µL	8.8 µLx
MgCl2	1.2 µL	26.4 µLx
Dream Taq Polymerase	0.1 µL	2.2 µL
fw Primer VF2	0.6 µL	13.2 µL
rv Primer VR	0.6 µL	13.2 µL
H2O ad 20 µL	15.1	332.2 µL x

The cPCR programme was as follows:

Temperature	Time [min]
98 °C	3:00
98 °C	0:30
55 °C	0:30
72 °C	2:00
go to step to (30x)
72 °C	5:00
4 °C	∞

The plate from the 1.10.2018 had just one grown colony which was picked, the plate from yesterday hat a lot more colonies, of which 18 were picked.

No clone was positive, a new restriction is necessary.

181003 – 5

Preparation for the activation assay of ldha

The part used for the activation assay was pSB1C3-BBa_K206000-BBa_0034-ldha.

Preparation of 3 x 200 mL cultures.

Medium	Inoculated with	Arabinose-Induction	OD at point of induction
200ml LB plus cAMP	180703LP02	2ml (0.1mol/L)	1
200ml LB plus cAMP	180703LP02	-	-
200ml LB	DH5a	-	-

Samples were centrifuged (20 min, 5000 rpm, 4 °C) to get a pellet. This pellet was stored at – 20 °C.

181003 – 6

PCR of synthetic DNA RNAG120-MlcRE

This was done in prospect to further experiments of the restriction for the next day.

PCR preparation according to the following table:

10x Dream Taq Buffer	2 µL
dNTPs 10 mM	0.4 µL
MgCl2	1.2 µL
Dream Taq Polymerase	0.1 µL
fw Primer VF2	0.6 µL
rv Primer VR	0.6 µL
H2O ad 20 µL	15.1 µL

PCR Programm as follows:

Temperature	Zeit [min]
95 °C	3:00
95 °C	0:30
55 °C	0:30
72 °C	0:45
go to step to (30x)
72 °C	5:00
4 °C	∞

Purification was done using the PCR purification kit following the protocol. The final concentration was measured with Nanodrop:

Lot number: 181003NiF01 with the concentration of 82.666 ng/µL.

Restriction, Ligation and transformation of pSB1C3-NOT-MlcRE-E0240

Restriction:

	Tube	1	2	3
	Part	RNAG120-MlcRE	pSB1K3	pSB1C3-E0240
	Lot#	181003NiF01	backbone	180925NaF02
	ρ	83 ng/µL	25 ng/µL	145 ng/µL
	Use	single insert	vector	vector
	target m	100 ng	200 ng	1000 ng

Volumes	DNA	1.21 µL	8.00 µL	6.91 µL
	NEBuffer 2.1	1.00 µL	2.00 µL	1.00 µL
	EcoRI-HF	0.50 µL	0.50 µL
	XbaI			0.50 µL
	SpeI	0.50 µL
	PstI		0.50 µL	0.50 µL
	FastAP		0.50 µL	0.50 µL
	H2O	6.79 µL	8.50 µL	0.59 µL
	ad	10.00 µL	20.00 µL	10.00 µL

	Länge Produkt	546	2070	878

	ρ final	10 ng/µL	13 ng/µL	2 ng/µL

Ligation:

Tube		L1
Product		pSB1K3-RNAG120-MlcRE-pSB1C3-E0240

Vector	Vector	pSB1K3
	Tube	2
	ρ	13 ng/µL
	Länge	2070 bp
	V	1.00 µL
Part 1	Part 1	RNAG120-MlcRE
	Tube	1
	ρ	10 ng/µL
	Länge	546 bp
	V	3.35 µL
Part 2	Part 2	pSB1C3-E0240
	Tube	3
	ρ	2 ng/µL
	Länge	878 bp
	V	24.75 µL
T4 Buffer		4.00 µL
T4 Ligase		0.50 µL
H2O		6.41 µL
ad		40.00 µL

Restriction, Ligation and transformation of pSB1K3-pBAD-BjalT-OmpT-FLAG-HlyA

Restriction:

Tube	27	28
Part	pGGA-pBAD-BjalT-OmpT-FLAG-HlyA	pSB1K3
Lot#	181003NB04	backbone
ρ	267 ng/µL	25 ng/µL
Use	vector	vector
target m	1000 ng	100 ng

DNA	7.49 µL	4.00 µL
NEBuffer 2.1	1.00 µL	1.00 µL
EcoRI-HF	0.50 µL	0.50 µL
XbaI
SpeI	0.50 µL	0.50 µL
PstI
FastAP		0.50 µL
H2O	0.51 µL	3.50 µL
ad	10.00 µL	10.00 µL

Länge Produkt	602	2070

ρ final	Gelex	10 ng/µL

Ligation

Tube		L23
Product		pSB1K3-pBAD-BjalT-OmpT-FLAG-HlyA

Vector	Vector	pSB1K3
	Tube	28
	ρ	10 ng/µL
	Länge	2070 bp
	V	2.56 µL
Part 1	Part 1	pGGA-pBAD-BjalT-OmpT-FLAG-HlyA
	Tube	27
	ρ	Gelex
	Länge	602 bp
	V
Part 2	Part 2
	Tube
	ρ
	Länge
	V
T4 Buffer		1.00 µL
T4 Ligase		0.50 µL
H2O		5.94 µL
ad		10.00 µL

Digestion test

LOT Number	Part	Test restriction number	Einschicken
181003NB03	pSB1C3-MlcRE	1	ja (mit vr)
181003NB05	psB1C3_accC #10	2	ja (vf2)
181003NB06	pSB1C3-oHybB-BBa_E0240	3	keine Bande
181003NB07	pSB1C3-cspD	4	ja(aber wahrscheinlich was anderes)
181003NB08	pGGA-NotMlcRE-HlyB-HlyD-TolC-OmpT	5

compound	volume
DNA	5 µL
EcoRI	0.5 µL
FastAP	0.5 µL
Buffer 2.1	1 µL
H2O	3 µL

Characterization of ldhA

According to protocol “ldhA Charakterisierung”.

Preparation of the buffer containing 0.5. mM EDTA and 1 mg/ml lysozyme for 40 mL:

5.8 mg EDTA
40 mg Lysozym
40 ml ddH20

Preparation of the cell lysates

Resuspension of the cell pellets in 2 mL buffer
Incubation for 1 hour on ice

Activity assay

Mastermix:

1300 µL Na-Pyrophosphate buffer
100 µL NAD+
200 µL INT
200 µL PMS

100 µL 40% Lactic acid solution (0.0013 M)
100 µL cell lysate (1000 µg protein)

Measurement

3 min in 20sec Abständen messen

Results:

DH5 alpha 1000 µg		ldhA + 1000 µg		ldhA- 1000 µg
Time	Durchschnittliche Extinktion	time	Durchschnitt Extinktion	time	Extinktion
0	0	0	0	0	0
20	1.14525	20	0.379	20	0.616
40	1.264	40	0.426	40	0.681
60	1.283	60	0.459	60	0.721
80	1.31925	80	0.484	80	0.7513333333
100	1.3325	100	0.504	100	0.7803333333
120	1.35375	120	0.524	120	0.585
140	1.36475	140	0.538	140	0.8676666667
160	1.3845	160	0.552	160	0.8496666667
180	1.28225	180	0.566	180	0.853

Minipreps of 180518BK15, 181001NB02

PCR of constructs from 03.10.2018 and L1 from 02.10.2018:

Tube

L1

L2

L3

L4

L5

L6

L7

L8

L9

L10

L11

L12

L13

L14

L15

L16

L17

L18

L19

L20

L21

L22

L23

L24

L25

L26

Product

pSB1C3_RNAG120_MlcRE-pSB1C3-E0240

pSB1C3-E0240-BBa_J45503

pSB1C3-oHybB-34-AOX1

pSB1C3-mraz

pSB1C3-BBa_K206000-32sulA

pSB1C3-BBa_K206000-34sulA

pSB1C3-BBa_B0032-cspD

pSB1C3-34-AOX1

pSB1C3-I13453-34-AOX1

pSB1C3-I13453-pSB1C3-BBa_B0032_cbtA

pSB1C3-oHybB-pSB1C3-E0240

pSB1C3_RNAG120_MlcRE-pSB1C3-BBa_I732018

pSB1C3-cspD

pSB1C3-accD

pSB1C3-ldhA

pSB1C3-ompT

pSB1C3-RNAG120

pSB1C3-NotMlcRE-HlyB-HlyD-TolC-OmpT

pSB1C3-pBAD-BjalT-OmpT-FLAG-HlyA

pSB1K3-NotMlcRE-HlyB-HlyD-TolC-OmpT

pSB1K3-pBAD-BjalT-OmpT-FLAG-HlyA

pSB1A3-NotMlcRE-HlyB-HlyD-TolC-OmpT

pSB1A3-pBAD-BjalT-OmpT-FLAG-HlyA

Vector

Vector

pSB1C3_RNAG120_MlcRE

pSB1C3-E0240

pSB1C3-oHybB

pSB1C3

pSB1C3-BBa_K206000

pSB1C3-BBa_B0032

pSB1C3

pSB1C3-I13453

pSB1C3-oHybB

pSB1C3_RNAG120_MlcRE

pSB1C3

pSB1K3

pSB1A3-BBa_B0034

Tube

1

3

5

7

9

14

15

16

5

1

7

29

23

28

24

30

ρ

Gelex

5 ng/µL

30 ng/µL

33 ng/µL

8 ng/µL

50 ng/µL

8 ng/µL

5 ng/µL

Gelex

30 ng/µL

14 ng/µL

19 ng/µL

16 ng/µL

26 ng/µL

Länge

2697 bp

2200 bp

2070 bp

2200 bp

2697 bp

2070 bp

V

2.90 µL

0.61 µL

4.25 µL

0.00 µL

0.29 µL

1.92 µL

4.45 µL

3.75 µL

1.62 µL

0.62 µL

0.58 µL

0.60 µL

2.13 µL

0.10 µL

1.83 µL

0.16 µL

1.60 µL

0.19 µL

0.98 µL

Part 1

Part 1

pSB1C3-E0240

BBa_J45503

34-AOX1

mraz

32sulA

34sulA

cspD

34-AOX1

pSB1C3-BBa_B0032_cbtA

pSB1C3-E0240

pSB1C3-BBa_I732018

cspD

accD

ldhA

ompT

RNAG120

pGGA-NotMlcRE-HlyB-HlyD-TolC-OmpT

pGGA-pBAD-BjalT-OmpT-FLAG-HlyA

pGGA-NotMlcRE-HlyB-HlyD-TolC-OmpT

pGGA-pBAD-BjalT-OmpT-FLAG-HlyA

pGGA-NotMlcRE-HlyB-HlyD-TolC-OmpT

pGGA-pBAD-BjalT-OmpT-FLAG-HlyA

Tube

2

4

6

8

11

12

13

6

17

2

18

13

19

20

21

22

25

26

27

25

26

27

25

26

27

ρ

17 ng/µL

Gelex

10 ng/µL

Gelex

8 ng/µL

17 ng/µL

7 ng/µL

10 ng/µL

Gelex

11 ng/µL

Gelex

11 ng/µL

Gelex

11 ng/µL

Länge

878 bp

1200 bp

222 bp

1200 bp

394 bp

878 bp

252 bp

222 bp

915 bp

987 bp

954 bp

120 bp

6500 bp

602 bp

6500 bp

602 bp

6500 bp

602 bp

V

4.54 µL

10.00 µL

8.54 µL

4.75 µL

7.20 µL

5.17 µL

8.16 µL

8.27 µL

8.22 µL

3.67 µL

6.88 µL

Part 2

Part 2

Tube

ρ

Länge

V

T4 Buffer

1.00 µL

2.00 µL

1.00 µL

2.00 µL

1.00 µL

2.00 µL

1.00 µL

2.00 µL

1.00 µL

2.00 µL

1.00 µL

T4 Ligase

0.50 µL

H2O

3.96 µL

8.50 µL

5.60 µL

6.89 µL

5.63 µL

7.50 µL

8.21 µL

6.58 µL

4.51 µL

0.00 µL

1.30 µL

1.70 µL

8.72 µL

8.65 µL

8.68 µL

2.70 µL

8.40 µL

8.79 µL

8.34 µL

0.02 µL

8.31 µL

0.64 µL

ad

10.00 µL

20.00 µL

10.00 µL

20.00 µL

10.00 µL

20.00 µL

10.00 µL

20.00 µL

10.00 µL

20.00 µL

10.00 µL

Tube

L1

L2

L3

L4

L5

L6

L7

L8

L9

L10

L11

L12

L13

L14

L15

L16

L17

L18

L19

L20

L21

L22

L23

L24

L25

L26

Product

pSB1C3_RNAG120_MlcRE-E0240

pSB1C3-E0240-BBa_J45503

pSB1C3-oHybB-34-AOX1

pSB1C3-mraz

pSB1C3-BBa_K206000-32sulA

pSB1C3-BBa_K206000-34sulA

pSB1C3-BBa_B0032-cspD

pSB1C3-34-AOX1

pSB1C3-I13453-34-AOX1

pSB1C3-I13453-pSB1C3-BBa_B0032_cbtA

pSB1C3-oHybB-E0240

pSB1C3_RNAG120_MlcRE-pSB1C3-BBa_I732018

pSB1C3-cspD

pSB1C3-accD

pSB1C3-ldhA

pSB1C3-ompT

pSB1C3-RNAG120

pSB1C3-NotMlcRE-HlyB-HlyD-TolC-OmpT

pSB1C3-pBAD-BjalT-OmpT-FLAG-HlyA

pSB1K3-NotMlcRE-HlyB-HlyD-TolC-OmpT

pSB1K3-pBAD-BjalT-OmpT-FLAG-HlyA

pSB1A3-NotMlcRE-HlyB-HlyD-TolC-OmpT

pSB1A3-pBAD-BjalT-OmpT-FLAG-HlyA

Vector

Vector

pSB1C3_RNAG120_MlcRE

pSB1C3-E0240

pSB1C3-oHybB

pSB1C3

pSB1C3-BBa_K206000

pSB1C3-BBa_B0032

pSB1C3

pSB1C3-I13453

pSB1C3-oHybB

pSB1C3_RNAG120_MlcRE

pSB1C3

pSB1K3

pSB1A3-BBa_B0034

Tube

1

3

5

7

9

14

15

16

5

1

7

23

24

ρ

7 ng/µL

Gelex

50 ng/µL

6 ng/µL

8 ng/µL

50 ng/µL

7 ng/µL

Gelex

7 ng/µL

50 ng/µL

33 ng/µL

Gelex

Länge

2697 bp

2200 bp

2070 bp

2200 bp

2697 bp

2070 bp

V

3.15 µL

0.61 µL

4.25 µL

0.00 µL

0.29 µL

2.21 µL

5.12 µL

6.93 µL

0.97 µL

0.37 µL

0.35 µL

0.36 µL

1.27 µL

0.06 µL

0.12 µL

1.02 µL

0.19 µL

1.54 µL

Part 1

Part 1

pSB1C3-E0240

BBa_J45503

34-AOX1

mraz

32sulA

34sulA

cspD

34-AOX1

pSB1C3-BBa_B0032_cbtA

pSB1C3-E0240

pSB1C3-BBa_I732018

cspD

accD

ldhA

ompT

RNAG120

pGGA-NotMlcRE-HlyB-HlyD-TolC-OmpT

pGGA-pBAD-BjalT-OmpT-FLAG-HlyA

pGGA-NotMlcRE-HlyB-HlyD-TolC-OmpT

pGGA-pBAD-BjalT-OmpT-FLAG-HlyA

pGGA-NotMlcRE-HlyB-HlyD-TolC-OmpT

pGGA-pBAD-BjalT-OmpT-FLAG-HlyA

Tube

2

4

6

8

11

12

13

6

17

2

18

13

19

20

21

22

25

26

27

25

26

27

25

26

27

ρ

6 ng/µL

Gelex

3 ng/µL

10 ng/µL

3 ng/µL

50 ng/µL

6 ng/µL

100 ng/µL

10 ng/µL

Gelex

Länge

878 bp

1200 bp

222 bp

1200 bp

394 bp

878 bp

252 bp

222 bp

915 bp

987 bp

954 bp

120 bp

6500 bp

602 bp

6500 bp

602 bp

6500 bp

602 bp

V

6.00 µL

33.80 µL

10.00 µL

34.12 µL

33.80 µL

1.28 µL

12.97 µL

0.48 µL

5.17 µL

8.16 µL

8.27 µL

8.22 µL

3.67 µL

Part 2

Part 2

Tube

ρ

Länge

V

T4 Buffer

2.00 µL

1.00 µL

4.00 µL

2.00 µL

1.00 µL

2.00 µL

4.00 µL

5.00 µL

1.00 µL

2.00 µL

1.00 µL

2.00 µL

1.00 µL

T4 Ligase

0.50 µL

H2O

8.35 µL

8.50 µL

1.70 µL

6.89 µL

5.63 µL

7.50 µL

1.09 µL

8.48 µL

2.10 µL

4.53 µL

1.09 µL

2.36 µL

-0.02 µL

8.89 µL

-0.07 µL

3.56 µL

8.44 µL

8.38 µL

7.48 µL

8.31 µL

6.96 µL

8.50 µL

ad

20.00 µL

10.00 µL

40.00 µL

20.00 µL

10.00 µL

20.00 µL

40.00 µL

50.00 µL

10.00 µL

20.00 µL

10.00 µL

20.00 µL

10.00 µL

Mastermix cPCR:

	1x	60 x
10x Dream Taq Buffer	2 µL	120 µL
dNTPs 10 mM	0.4 µL	24 µL
MgCl2	1.2 µL	72 µL
Dream Taq Polymerase	0.1 µL	6 µL
fw Primer VF2	0.6	36 µL
rv Primer VR	0.6	36 µL
H2O ad 20 µL	15.1	906 µL

Restriction

	Tube	1	2	3
	Part	pSB1C3	pBAD-HlyB-HlyD-TolC-OmpT	pBAD-HlyB-HlyD-TolC-OmpT
	Lot#	aus Kit	R	P
	ρ	25 ng/µL	15 ng/µL	10 ng/µL
	Use
	target m

Volumes	DNA	4.00 µL	7.00 µL	7.00 µL
	NEBuffer 2.1	1.00 µL	1.00 µL	1.00 µL
	EcoRI-HF	0.50 µL	0.50 µL	0.50 µL
	XbaI
	SpeI
	PstI	0.50 µL	0.50 µL	0.50 µL
	FastAP	0.50 µL
	H2O	3.50 µL	1.00 µL	1.00 µL
	ad	10.00 µL	10.00 µL	10.00 µL

	Länge Produkt	2000	6300	6300

	ρ final	10 ng/µL	11 ng/µL	7 ng/µL

Ligation

Tube		L1	L2
Product		pSB1C3-pBAD-HlyB-HlyD-TolC-OmpT	pSB1C3-pBAD-HlyB-HlyD-TolC-OmpT

Vector	Vector	pSB1C3	pSB1C3
	Tube	1	1
	ρ	10 ng/µL	10 ng/µL
	Länge	2000 bp	2000 bp
	V	0.50 µL	0.50 µL
Part 1	Part 1	pBAD-HlyB-HlyD-TolC-OmpT	pBAD-HlyB-HlyD-TolC-OmpT
	Tube	2	3
	ρ	11 ng/µL	7 ng/µL
	Länge	6300 bp	6300 bp
	V	8.00 µL	8.00 µL
Part 2	Part 2
	Tube
	ρ
	Länge
	V
T4 Buffer		1.00 µL	1.00 µL
T4 Ligase		0.50 µL	0.50 µL
H2O		0.00 µL	0.00 µL
ad		10.00 µL	10.00 µL

Restriction/Ligation/Trafo

Restriction:

Tube

1

2

3

4

5

6

7

8

9

10

11

12

13

14

15

16

17

18

19

20

21

22

23

24

25

26

27

28

29

30

31

32

33

Part

pSB1C3_RNAG120_MlcRE

pSB1C3-E0240

BBa_J45503

pSB1C3-oHybB

34-AOX1

pSB1C3

mraz

pSB1C3-BBa_K206000

31sulA

32sulA

34sulA

cspD

pSB1C3-BBa_B0032

pSB1C3

pSB1C3-I13453

pSB1C3-BBa_B0032_cbtA

pSB1C3-BBa_I732018

accD

ldhA

ompT

RNAG120

pSB1K3

pSB1A3

pGGA-NotMlcRE-HlyB-HlyD-TolC-OmpT

pGGA-pBAD-BjalT-OmpT-FLAG-HlyA

pSB1K3

pSB1C3

pSB1A3

RNAG120-MlcRE

pSB1K3

pSB1C3-E0240

Lot#

180925NF01

180925NF02

180817LD10

180625NiF12

181001NB05

180920LP01

backbone

180622NB08

neue Miniprep

180905AW03

180622NB04

neue Miniprep

180924AW1

180925NaF04

180926NaF03

181004NF01

180921BK09

180622NB01

180827LP01

180909BK09

backbone

181003NB08

180920OM02

181003NB04

backbone

181003NiF01

backbone

180925NaF02

ρ

140 ng/µL

144.673

200 ng/µL

45 ng/µL

107 ng/µL

575 ng/µL

25 ng/µL

44.175

50 ng/µL

28 ng/µL

40 ng/µL

43 ng/µL

50 ng/µL

81 ng/µL

95 ng/µL

126 ng/µL

93.011

93 ng/µL

213 ng/µL

57 ng/µL

68 ng/µL

77 ng/µL

25 ng/µL

25

184 ng/µL

800 ng/µL

6 ng/µL

25 ng/µL

25

83 ng/µL

25 ng/µL

145 ng/µL

Use

Vector

vector

single insert

vector

single insert

vector

single insert

vector

single insert

vector

single insert

vector

single insert

vector

target m

1000 ng

100 ng

1000 ng

100 ng

1000 ng

100 ng

1000 ng

100 ng

200 ng

1000 ng

100 ng

1000 ng

Volumes

DNA

7.15 µL

6.91 µL

5.00 µL

2.24 µL

9.34 µL

0.17 µL

4.00 µL

2.26 µL

20.20 µL

3.57 µL

2.50 µL

2.33 µL

2.00 µL

12.42 µL

10.57 µL

7.93 µL

10.75 µL

21.43 µL

0.47 µL

1.77 µL

1.47 µL

1.30 µL

8.00 µL

10.90 µL

2.50 µL

15.00 µL

4.00 µL

1.21 µL

4.00 µL

6.91 µL

NEBuffer 2.1

1.00 µL

2.00 µL

4.00 µL

1.00 µL

3.00 µL

1.00 µL

2.00 µL

3.00 µL

1.00 µL

2.00 µL

1.00 µL

2.00 µL

1.00 µL

EcoRI-HF

0.50 µL

XbaI

0.50 µL

SpeI

0.50 µL

PstI

0.50 µL

FastAP

0.50 µL

H2O

0.85 µL

0.59 µL

2.50 µL

5.26 µL

7.66 µL

34.83 µL

3.50 µL

5.74 µL

5.30 µL

4.43 µL

5.50 µL

5.67 µL

6.00 µL

4.08 µL

5.93 µL

8.57 µL

6.25 µL

4.57 µL

7.53 µL

6.23 µL

6.53 µL

6.70 µL

8.50 µL

6.10 µL

5.50 µL

2.00 µL

3.50 µL

6.79 µL

3.50 µL

1.09 µL

ad

10.00 µL

20.00 µL

40.00 µL

10.00 µL

30.00 µL

10.00 µL

20.00 µL

30.00 µL

10.00 µL

20.00 µL

10.00 µL

20.00 µL

10.00 µL

Länge Produkt

2697 bp

878 bp

2200 bp

1200 bp

2070

222

2070

2200

394

252

915

987

954

120

2070

6500

602

2070

546

2070

878

ρ final

Gelex

8 ng/µL

Gelex

20 ng/µL

10 ng/µL

33 ng/µL

10 ng/µL

8 ng/µL

50 ng/µL

Gelex

11 ng/µL

10 ng/µL

PCR Purification

Gelex

6 ng/µL

5 ng/µL

10 ng/µL

2 ng/µL

Gelex

Ligation

Tube

L1

L2

L3

L4

L5

L6

L7

L8

L9

L10

L11

L12

L13

L14

L15

L16

L17

L18

L19

L20

L21

L22

L23

L24

L25

L26

L27

Product

pSB1C3_RNAG120_MlcRE-pSB1C3-E0240

pSB1C3-E0240-BBa_J45503

pSB1C3-oHybB-34-AOX1

pSB1C3-mraz

pSB1C3-BBa_K206000-32sulA

pSB1C3-BBa_K206000-34sulA

pSB1C3-BBa_B0032-cspD

pSB1C3-34-AOX1

pSB1C3-I13453-34-AOX1

pSB1C3-I13453-pSB1C3-BBa_B0032_cbtA

pSB1C3-oHybB-pSB1C3-E0240

pSB1C3_RNAG120_MlcRE-pSB1C3-BBa_I732018

pSB1C3-cspD

pSB1C3-accD

pSB1C3-ldhA

pSB1C3-ompT

pSB1C3-RNAG120

pSB1C3-NotMlcRE-HlyB-HlyD-TolC-OmpT

pSB1C3-pBAD-BjalT-OmpT-FLAG-HlyA

pSB1K3-NotMlcRE-HlyB-HlyD-TolC-OmpT

pSB1K3-pBAD-BjalT-OmpT-FLAG-HlyA

pSB1A3-NotMlcRE-HlyB-HlyD-TolC-OmpT

pSB1A3-pBAD-BjalT-OmpT-FLAG-HlyA

pSB1K3-RNAG120-MlcRE-pSB1C3-E0240

Vector

Vector

pSB1C3_RNAG120_MlcRE

pSB1C3-E0240

pSB1C3-oHybB

pSB1C3

pSB1C3-BBa_K206000

pSB1C3-BBa_B0032

pSB1C3

pSB1C3-I13453

pSB1C3-oHybB

pSB1C3_RNAG120_MlcRE

pSB1C3

pSB1K3

pSB1A3

pSB1K3

Tube

1

3

5

7

9

14

15

16

5

1

7

29

23

28

24

30

32

ρ

Gelex

8 ng/µL

20 ng/µL

33 ng/µL

8 ng/µL

50 ng/µL

Gelex

8 ng/µL

Gelex

20 ng/µL

5 ng/µL

PCR Purification

5 ng/µL

Gelex

5 ng/µL

2 ng/µL

Länge

2697 bp

2200 bp

2070 bp

2200 bp

2697 bp

2070 bp

V

1.93 µL

0.61 µL

4.25 µL

0.00 µL

0.29 µL

2.50 µL

2.46 µL

0.94 µL

0.88 µL

0.91 µL

3.22 µL

0.16 µL

4.90 µL

5.51 µL

5.32 µL

5.50 µL

Part 1

Part 1

pSB1C3-E0240

BBa_J45503

34-AOX1

mraz

32sulA

34sulA

cspD

34-AOX1

pSB1C3-BBa_B0032_cbtA

pSB1C3-E0240

pSB1C3-BBa_I732018

cspD

accD

ldhA

ompT

RNAG120

pGGA-NotMlcRE-HlyB-HlyD-TolC-OmpT

pGGA-pBAD-BjalT-OmpT-FLAG-HlyA

pGGA-NotMlcRE-HlyB-HlyD-TolC-OmpT

pGGA-pBAD-BjalT-OmpT-FLAG-HlyA

pGGA-NotMlcRE-HlyB-HlyD-TolC-OmpT

pGGA-pBAD-BjalT-OmpT-FLAG-HlyA

RNAG120-MlcRE

Tube

2

4

6

8

11

12

13

6

17

2

18

13

19

20

21

22

25

26

27

25

26

27

25

26

27

31

ρ

Gelex

10 ng/µL

Gelex

11 ng/µL

10 ng/µL

Gelex

6 ng/µL

Gelex

6 ng/µL

Gelex

6 ng/µL

10 ng/µL

Länge

878 bp

1200 bp

222 bp

1200 bp

394 bp

878 bp

252 bp

222 bp

915 bp

987 bp

954 bp

120 bp

6500 bp

602 bp

6500 bp

602 bp

6500 bp

602 bp

546 bp

V

10.00 µL

4.30 µL

5.17 µL

8.16 µL

8.27 µL

8.22 µL

3.67 µL

6.00 µL

3.35 µL

Part 2

Part 2

pSB1C3-E0240

Tube

33

ρ

Gelex

Länge

878 bp

V

T4 Buffer

1.00 µL

2.00 µL

1.00 µL

2.00 µL

1.00 µL

2.00 µL

1.00 µL

2.00 µL

1.00 µL

2.00 µL

1.00 µL

2.00 µL

T4 Ligase

0.50 µL

H2O

8.50 µL

6.57 µL

6.89 µL

5.63 µL

7.50 µL

8.21 µL

8.50 µL

6.00 µL

4.20 µL

0.87 µL

8.41 µL

8.35 µL

8.37 µL

1.61 µL

8.34 µL

6.60 µL

8.50 µL

5.99 µL

8.50 µL

6.18 µL

8.65 µL

ad

10.00 µL

20.00 µL

10.00 µL

20.00 µL

10.00 µL

20.00 µL

10.00 µL

20.00 µL

10.00 µL

20.00 µL

10.00 µL

20.00 µL

Characterization of AOX1

Two Cultures of 100 mL were prepared. For the culture containing pBAD-AOX1, chloramphenicol (1:1000) and arabinose (1 mL of 0.1 mol/L) was added. A starter culture of each 3 mL overnight culture was transferred into the according media at an OD of 0.01 and grown to an OD of 0.2. After 120 min wt DH5alpha at OD 0.3 and pBAD-AOX1 cultures were transferred into an incubator at 15 °C and measured every half hour.

1 x 100 mL pBAD-AOX1 (induziert):

Time [min]	OD	Temperature [°C]
0	0.01	37
60	0.02	37
120	0.26	37
150	0.27	18.4
180	0.27	16.1
210	0.28	18.0
240	0.31	17.4
270	0.33	17.6
300	0.37	16.8
330
360
390
420

1 x 100 mL DH5alpha:

Time [min]	OD	Temperature [°C]
0	0.01	37
60	0.02	37
120	0.34	37
150	0.54	18.1
180	0.64	15.7
210	0.69	17.7
240	0.71	17.2
270	0.73	17.5
300	0.88	16.8
330
360
390
420

Minipreps and cryostocks of Ligations from 1801004

Cryostocks were prepared for the following samples:

Lot No.	Part
181005AK01	pSB1C3-accD #3
181005AK02	pSB1C3-ompT #4
181005AK03	pSB1C3-ompT #5
181005AK04	pSB1C3- NOT-MlCRE-E0240 #1
181005AK05	pSB1C3- NOT-MlCRE-E0240 #3
181005AK06	pSB1C3- NOT-MlCRE-E0240 #4
181005AK07	pSB1C3- NOT-MlCRE-E0240 #5
181005AK08	pSB1C3- NOT-MlCRE-E0240 #6
181005AK09	pSB1C3- NOT-MlCRE-E0240 #7
181005AK10	pSB1C3- NOT-MlCRE-E0240 #8
181005AK11	pSB1C3-oHybB-E0240 #1
181005AK12	pSB1C3-oHybB-E0240 #3
181005AK13	pSB1C3-cspD #1
181005AK14	pSB1C3-cspD #2
181005AK15	pSB1C3-cspD #5

After revision of the agarose gel from 181004, the samples marked in grey were discarded.

Set up the cPCRs from 181004

The transformed cultures of the ligations from 181004:

L1 (pSB1C3-pBAD-HlyB-HlyD-TolC-OmpT)

L2 (pSB1C3-pBAD-HlyB-HlyD-TolC-OmpT)

L23 (pSB1C3-pBAD-BjalT-OmpT-FLAG-HlyA)

L17 (pSB1C3-RNAG120)

Plate from 181003 where 2 colonies grew (pSB1K3-pBAD-BjalT-ompT-FLAg-HlyA)

Mastermix:

	1x	40 x
10x Dream Taq Buffer	2 µL	80 µL
dNTPs 10 mM	0.4 µL	16 µL
MgCl2	1.2 µL	48 µL
Dream Taq Polymerase	0.1 µL	4 µL
fw Primer VF2	0.6	24 µL
rv Primer VR	0.6	24 µL
H2O ad 20 µL	15.1	604 µL

PCR program:

Temperature	Time
98 °C	3:00
98 °C	0:30
55 °C	0:30
72 °C	3:00
go to step 2 30x
72 °C	5:00
4 °C	∞

PCRs of pSB1C3

To control the PCR preparation two PCR samples were prepared, with one of them using the Phusion HF and the other using Q5 Polymerase.

Mastermix:

Sample 1
5x Phusion HF	10 µL
dNTPs 10 mM	1 µL
SB-prep-3P1	0.35 µL
SB-prep-2Ea	0.35 µL
Template (181001NB04)	0.5
Phusion Polymerase	0.5
H2O ad 20 µL	37.3

Sample 2
Q5 2x Mastermix	10 µL
SB-prep-3P1	0.2 µL
SB-prep-2Ea	0.2 µL
Template (181001NB04)	0.5 µL
H2O ad 20 µL	37.3

PCR program:

Temperature	Time
98 °C	3:00
98 °C	0:30
55 °C	0:30
72 °C	2:00
go to step 2 30x
72 °C	10:00
4 °C	∞

Restriction

	Tube	7	13	18	19	21
	Part	pSB1C3	cspD	pSB1C3-BBa_I732018	accD	ompT
	Lot#	181001NB06	181001NB01	181004NF01	180921BK09	180827LP01
	ρ	25 ng/µL	37 ng/µL	93 ng/µL	213 ng/µL	68 ng/µL
	Use	vector	single insert	vector	single insert	single insert
	target m	200 ng	100 ng	1000 ng	100 ng	100 ng

Volumes	DNA	8.00 µL	2.73 µL	10.72 µL	0.47 µL	1.47 µL
	NEBuffer 2.1	2.00 µL	1.00 µL	2.00 µL	1.00 µL	1.00 µL
	EcoRI-HF	0.50 µL	0.50 µL		0.50 µL	0.50 µL
	XbaI			0.50 µL
	SpeI
	PstI	0.50 µL	0.50 µL	0.50 µL	0.50 µL	0.50 µL
	FastAP	0.50 µL
	H2O	8.50 µL	5.27 µL	6.28 µL	7.53 µL	6.53 µL
	ad	20.00 µL	10.00 µL	20.00 µL	10.00 µL	10.00 µL

	Länge Produkt	2070	222	252	915	954

	ρ final	10 ng/µL	10 ng/µL	2 ng/µL	10 ng/µL	10 ng/µL

Ligation

Tube

L1

L2

L3

L4

L5

L6

L7

L8

L9

L10

L11

L12

L13

L14

L15

L16

L17

L18

L19

L20

L21

L22

L23

L24

L25

L26

L27

L28

L29

L30

L31

Product

pSB1C3_RNAG120_MlcRE-pSB1C3-E0240

pSB1C3-E0240-BBa_J45503

pSB1C3-oHybB-34-AOX1

pSB1C3-mraz

pSB1C3-BBa_K206000-32sulA

pSB1C3-BBa_K206000-34sulA

pSB1C3-BBa_B0032-cspD

pSB1C3-34-AOX1

pSB1C3-I13453-34-AOX1

pSB1C3-I13453-pSB1C3-BBa_B0032_cbtA

pSB1C3-oHybB-pSB1C3-E0240

pSB1C3_RNAG120_MlcRE-pSB1C3-BBa_I732018

pSB1C3-cspD

pSB1C3-accD

pSB1C3-ldhA

pSB1C3-ompT

pSB1C3-RNAG120

pSB1C3-NotMlcRE-HlyB-HlyD-TolC-OmpT

pSB1C3-pBAD-BjalT-OmpT-FLAG-HlyA

pSB1K3-NotMlcRE-HlyB-HlyD-TolC-OmpT

pSB1K3-pBAD-BjalT-OmpT-FLAG-HlyA

pSB1A3-NotMlcRE-HlyB-HlyD-TolC-OmpT

pSB1A3-pBAD-BjalT-OmpT-FLAG-HlyA

pSB1K3-RNAG120-MlcRE-pSB1C3-E0240

pSB1C3-RnaG120-Operator

pSB1K3-RNAG120-MlcRE-pSB1C3-BBa_B0034-ldhA

pSB1C3-RNAG120-MlcRE-pSB1C3-BBa_B0034-ldhA

pSB1C3-RNAG120-MlcRE-pSB1C3-E0240

Vector

Vector

pSB1C3_RNAG120_MlcRE

pSB1C3-E0240

pSB1C3-oHybB

pSB1C3

pSB1C3-BBa_K206000

pSB1C3-BBa_B0032

pSB1C3

pSB1C3-I13453

pSB1C3-oHybB

pSB1C3_RNAG120_MlcRE

pSB1C3

pSB1K3

pSB1A3

pSB1K3

pSB1C3

pSB1K3

pSB1C3

Tube

1

3

5

7

9

14

15

16

5

1

7

29

23

28

24

30

32

29

32

7

ρ

Gelex

10 ng/µL

33 ng/µL

8 ng/µL

50 ng/µL

Gelex

10 ng/µL

PCR Purification

3 ng/µL

PCR Purification

6 ng/µL

14 ng/µL

10 ng/µL

14 ng/µL

10 ng/µL

Länge

2697 bp

2200 bp

2070 bp

2200 bp

2697 bp

2070 bp

V

0.61 µL

4.25 µL

0.00 µL

0.29 µL

4.83 µL

1.84 µL

1.73 µL

1.78 µL

6.33 µL

0.31 µL

2.56 µL

9.18 µL

3.95 µL

0.92 µL

4.08 µL

0.86 µL

1.18 µL

1.27 µL

Part 1

Part 1

pSB1C3-E0240

BBa_J45503

34-AOX1

mraz

32sulA

34sulA

cspD

34-AOX1

pSB1C3-BBa_B0032_cbtA

pSB1C3-E0240

pSB1C3-BBa_I732018

cspD

accD

ldhA

ompT

RNAG120

pGGA-NotMlcRE-HlyB-HlyD-TolC-OmpT

pGGA-pBAD-BjalT-OmpT-FLAG-HlyA

pGGA-NotMlcRE-HlyB-HlyD-TolC-OmpT

pGGA-pBAD-BjalT-OmpT-FLAG-HlyA

pGGA-NotMlcRE-HlyB-HlyD-TolC-OmpT

pGGA-pBAD-BjalT-OmpT-FLAG-HlyA

RNAG120-MlcRE

RnaG120-Operator

RNAG120-MlcRE

Tube

2

4

6

8

11

12

13

6

17

2

18

13

19

20

21

22

25

26

27

25

26

27

25

26

27

31

35

31

ρ

Gelex

10 ng/µL

Gelex

2 ng/µL

10 ng/µL

Gelex

8 ng/µL

Gelex

8 ng/µL

Gelex

8 ng/µL

10 ng/µL

Länge

878 bp

1200 bp

222 bp

1200 bp

394 bp

878 bp

252 bp

222 bp

915 bp

987 bp

954 bp

120 bp

6500 bp

602 bp

6500 bp

602 bp

6500 bp

602 bp

546 bp

300 bp

546 bp

V

10.00 µL

21.34 µL

5.17 µL

8.16 µL

8.27 µL

8.22 µL

3.67 µL

9.79 µL

3.35 µL

5.92 µL

3.11 µL

3.35 µL

Part 2

Part 2

pSB1C3-E0240

pSB1C3-BBa_B0034-ldhA

pSB1C3-E0240

Tube

33

34

33

ρ

3 ng/µL

Gelex

3 ng/µL

Länge

878 bp

1000 bp

878 bp

V

17.42 µL

T4 Buffer

1.00 µL

2.00 µL

1.00 µL

2.00 µL

1.00 µL

3.00 µL

2.00 µL

1.00 µL

2.00 µL

1.00 µL

3.00 µL

1.00 µL

2.00 µL

3.00 µL

2.00 µL

1.00 µL

3.00 µL

T4 Ligase

0.50 µL

H2O

8.50 µL

6.89 µL

5.63 µL

7.50 µL

8.21 µL

8.50 µL

5.16 µL

7.50 µL

8.19 µL

5.15 µL

8.50 µL

7.52 µL

8.50 µL

3.76 µL

4.82 µL

7.50 µL

4.53 µL

4.20 µL

4.47 µL

ad

10.00 µL

20.00 µL

10.00 µL

20.00 µL

10.00 µL

30.00 µL

20.00 µL

10.00 µL

20.00 µL

10.00 µL

30.00 µL

10.00 µL

20.00 µL

30.00 µL

20.00 µL

10.00 µL

30.00 µL

Minipreps

The minipreps were performed on the ONC, the following Lot No. were given:

Lot No.	Part	Concentration [ng/µL]
181006LD01	pSB1C3-RnaG120-Operator + MlcRE	53.364
181006LD02	pSB1C3-E0240	64.174
181006LD03	pSB1C3-E0240	71.938
181006LD04	pGGA-pBAD-BjalT-OmpT-FLAG-HlyA	61.228
181006LD05	pGGA-pBAD-BjalT-OmpT-FLAG-HlyA	53.201
181006LD06	L1 #3 - pSB1C3_RNAG120_MlcRE-E0240	73.904
181006LD07	L20 #5 - pSB1C3-pBAD-BjalT-OmpT-FLAG-HlyA	72.132
181006LD08	L23#1 - pSB1K3-pBAD-BjalT-OmpT-FLAG-HlyA	38.318
181006LD09	L23 #5 - pSB1K3-pBAD-BjalT-OmpT-FLAG-HlyA	59.710

Characterization of AOX1

Preparation of Arabinose solution (0.1 M): To 10 mL H2O 0.15 g Arabinose (L+, Roth) were added and mixed.

Characterization of AOX1: Two Cultures of 100 mL were prepared. For the culture containing pBAD-AOX1, chloramphenicol (1:1000) and arabinose (1 mL of 0.1 mol/L) was added. A starter culture of each 5 mL overnight culture was transferred into the according media at an OD of 0.01 and grown to an OD of 0.2. After 120 min wt DH5alpha at OD 0.3 and pBAD-AOX1 cultures were transferred into an incubator at 15 °C and measured every half hour. 300 min after induction arabinose was given a second time.

1 x 100 mL pBAD-AOX1 (induced):

Time	OD	Temperature (°C)
0	0.01	37
60	0.138	37
120	0.264	37
150
180	0.389	21.2
210	0.399	18.2
240	0.366	17.5
270	0.409	17.5
300	0.430	17.5
330	0.451	17.5
360	0.482	17.5
390	0.701	17.5

1 x 100 mL DH5alpha:

Time	OD	Temperature (°C)
0	0.01	37
60	0.234	37
120	0.296 (nach Angleich)	37
150
180	0.768	19.9
210	0.831	17.1
240	0.996	17.0
270	1.069	17.2
300	1.416	17.0
330	1.611	17.2
360	1.648	17.3
390	4.409	17.2

cPCR performed with the transformation of the 181006 and 181004

Tubes	Part
01-03	pSB1C3-ompT
04-12	pSB1C3-RnaG120-MlcRE-E0240
13-18	pSB1C3-RnaG120operator
19-21	pSB1C3-pBAD-BjaIT-OmptSITE-FLAG
22-25	pSB1C3-cspD
26-30	pSB1C3-RnaG120-MlcRE-E0840
31-34	pSB1K3-pBAD-Bjait-OmpTsite-FLAG
35	pSB1A3-pBAD-Bjait-OmpTsite-FLAG
36-41	L1 Excretion GGA
42-48	L2 Excretion GGA

Mastermix cPCR:

	1x	45 x (for Parts in pSB1C3)	5 x (for others)
10x Dream Taq Buffer	2 µL	90 µL	10
dNTPs 10 mM	0.4 µL	18 µL	2
MgCl2	0.6 µL	27 µL	3
Dream Taq Polymerase	0.1 µL	4.5 µL	0.5
fw Primer VF2	0.6	27 µL
rv Primer VR	0.6	27 µL
H2O ad 20 µL	15.1	679 µL	75.5
BBSuf_Syn_r			3
BBPre_Syn_f			3

PCR Protocol:

Temperature	time	time (for Excretion GGA)
95 °C	3:00	3:00
94 °C	0:30	0:30
55 °C	0:30
72 °C	2:00	7:00
go to step to (30x)
72 °C	5:00	10:00
4 °C	∞	∞

PCR for Growth Inhibition with pGGA-dapAP-30cbtA-30cspD-30mraZ-34sulA

Mastermix:

	1x
5x Phusion Buffer	10 µL
dNTPs 10 mM	1 µL
Phusion Polymerase	0.5 µL
Primer P130Pre_34_cbtA_f	0.35 µL
Primer mraZ_Suf_r	0.35 µL
Template (181007NB06: pGGA-dapAP-30cbtA-30cspD-30mraZ-34sulA)	1 µL
H2O ad 20 µL	36.8 µL

PCR program:

Temperature	Time
95°C	3:00
94°C	0:30
55°C	0:30
72°C	1:30
go to step 2 30x
72°C	5:00
4°C	∞

cPCR was performed with the transformation of 181003 and 181004

Tubes	Part
1-2	pSB1K3-pBAD-BjaIT-OmptSITE-FLAG (L23)
3-7	pSB1K3-pBAD-BjaIT-OmptSITE-FLAG (L23)
8-15	pSB1C3-RnaG120 (L17)
16-23	pSB1C3-pBAD-BjaIT-OmptSITE-FLAG (L20)

Mastermix:

	1x	17 x (for Parts in pSB1C3)	8 x (for others)
10x Dream Taq Buffer	2 µL	34 µL	16 µL
dNTPs 10 mM	0.4 µL	6.8 µL	3.2 µL
MgCl2	0.6 µL	10.2 µL	4.8 µL
Dream Taq Polymerase	0.1 µL	1.7 µL	0.8 µL
fw Primer VF2	0.6	10.2 µL	-
rv Primer VR	0.6	10.2 µL	-
H2O ad 20 µL	15.7	266.9 µL	125.6
BBSuf_Syn_r	0.6	-	4.8 µL
BBPre_Syn_f	0.6	-	4.8 µL

PCR program:

Temperature	time
95 °C	3:00
94 °C	0:30
55 °C	0:30
72 °C	2:00
go to step to (30x)
72 °C	5:00
4 °C	∞

AOX1 Characterization

t [h]	T [°C]	OD 600 DH5a	T [°C]	OD 600 AOX1+Arabinose
0	-	0.012	-	0.004
2	34.5	0.021	34.5	0.007
3	34.5	0.089	36	0.044
4 (Nach Messung Schüttler auf 15°C gestellt)	37.1	0.416	36.6	0.242
5	18.5	0.367	18.2	0.349
6	16.5	0.47	16.2	0.384
7	16.5	0.577	16.7	0.443
8	16.5	0.711	16.5	0.445
9	16.5	0.576	16.7	0.474
10	16.8	0.667	16.7	0.493

Restriction

Tube

1

2

3

4

5

6

7

8

9

10

11

12

13

14

15

16

17

18

19

20

21

22

23

24

25

26

27

28

29

30

31

32

33

34

35

36

37

Part

pSB1C3_RNAG120_MlcRE

pSB1C3-E0240

BBa_J45503

pSB1C3-oHybB

34-AOX1

pSB1C3

mraz

pSB1C3-BBa_K206000

31sulA

32sulA

34sulA

cspD

pSB1C3-BBa_B0032

pSB1C3

pSB1C3-I13453

pSB1C3-BBa_B0032_cbtA

pSB1C3-BBa_I732018

accD

ldhA

ompT

RNAG120

pSB1K3

pSB1A3

pGGA-NotMlcRE-HlyB-HlyD-TolC-OmpT

pGGA-pBAD-BjalT-OmpT-FLAG-HlyA

pSB1K3

pSB1C3

pSB1A3

RNAG120-MlcRE

pSB1K3

pSB1C3-E0240

pSB1C3-BBa_B0034-ldhA

RnaG120-Operator

cbtA-cspD-mraZ

pSB1C3-BBa_I13453

Lot#

181006LD01

181006LD03

180817LD10

180625NiF12

181001NB05

180920LP01

181005NiF01

180622NB08

neue Miniprep

180905AW03

181001NB01

neue Miniprep

180924AW1

180925NaF04

180926NaF03

181004NF01

180921BK09

180622NB01

180827LP01

181003BK01

backbone

neue Miniprep

180920OM02

181006LD04

backbone

181005NiF01

backbone

181003NiF01

backbone

181006LD03

180426NK28

181003BK01

181008BK01

180925NaF04

ρ

53 ng/µL

71.938

200 ng/µL

45 ng/µL

107 ng/µL

575 ng/µL

146 ng/µL

44.175

50 ng/µL

28 ng/µL

40 ng/µL

43 ng/µL

37 ng/µL

81 ng/µL

95 ng/µL

126 ng/µL

93.011

93 ng/µL

213 ng/µL

57 ng/µL

68 ng/µL

21 ng/µL

25 ng/µL

25

800 ng/µL

2 ng/µL

25 ng/µL

146 ng/µL

25 ng/µL

83 ng/µL

25 ng/µL

71.938

86 ng/µL

21 ng/µL

28 ng/µL

126 ng/µL

Use

Vector

vector

single insert

vector

single insert

vector

single insert

vector

single insert

vector

single insert

vector

single insert

vector

single insert

target m

1000 ng

100 ng

1000 ng

100 ng

500 ng

100 ng

1000 ng

100 ng

1000 ng

100 ng

300 ng

200 ng

1000 ng

500 ng

100 ng

500 ng

100 ng

300 ng

200 ng

1000 ng

300 ng

1000 ng

Volumes

DNA

18.74 µL

13.90 µL

5.00 µL

2.24 µL

9.34 µL

0.17 µL

3.42 µL

2.26 µL

20.20 µL

3.57 µL

2.50 µL

2.33 µL

2.70 µL

12.42 µL

10.57 µL

7.93 µL

10.75 µL

10.72 µL

0.47 µL

1.77 µL

1.47 µL

14.29 µL

8.00 µL

1.25 µL

20.00 µL

4.00 µL

3.42 µL

4.00 µL

3.61 µL

8.00 µL

13.90 µL

11.69 µL

14.03 µL

10.55 µL

7.93 µL

NEBuffer 2.1

3.00 µL

2.00 µL

1.00 µL

2.00 µL

4.00 µL

1.00 µL

3.00 µL

1.00 µL

2.00 µL

1.00 µL

2.00 µL

1.00 µL

3.00 µL

1.00 µL

2.00 µL

EcoRI-HF

0.50 µL

XbaI

0.50 µL

SpeI

0.50 µL

PstI

0.50 µL

FastAP

0.50 µL

H2O

7.26 µL

2.60 µL

2.50 µL

5.76 µL

7.66 µL

34.83 µL

4.08 µL

5.74 µL

5.30 µL

4.43 µL

5.50 µL

5.67 µL

5.30 µL

4.08 µL

5.93 µL

8.57 µL

6.25 µL

6.28 µL

7.53 µL

6.23 µL

6.53 µL

2.71 µL

8.50 µL

8.00 µL

6.75 µL

6.00 µL

3.50 µL

4.08 µL

3.50 µL

4.39 µL

8.50 µL

2.60 µL

5.31 µL

2.97 µL

6.45 µL

8.57 µL

ad

30.00 µL

20.00 µL

10.00 µL

20.00 µL

40.00 µL

10.00 µL

30.00 µL

10.00 µL

20.00 µL

10.00 µL

20.00 µL

10.00 µL

30.00 µL

10.00 µL

20.00 µL

Länge Produkt

2697 bp

878 bp

2200 bp

1200 bp

2070

222

2070

2200

394

252

915

987

954

120

2070

6500

602

2070

546

2070

878

1000

300

2200

ρ final

Gelex

18 ng/µL

10 ng/µL

33 ng/µL

10 ng/µL

8 ng/µL

50 ng/µL

Gelex

10 ng/µL

15 ng/µL

PCR Purification

Gelex

2 ng/µL

19.149

PCR Purification

30 ng/µL

9 ng/µL

Gelex

4 ng/µL

15 ng/µL

6 ng/µL

PCR Purification

Gelex

PCR Purification

Gelex

Ligation

Tube

L1

L2

L3

L4

L5

L6

L7

L8

L9

L10

L11

L12

L13

L14

L15

L16

L17

L18

L19

L20

L21

L22

L23

L24

L25

L26

L27

L28

L29

L30

L31

L32

Product

pSB1C3_RNAG120_MlcRE-pSB1C3-E0240

pSB1C3-E0240-BBa_J45503

pSB1C3-oHybB-34-AOX1

pSB1C3-mraz

pSB1C3-BBa_K206000-32sulA

pSB1C3-BBa_K206000-34sulA

pSB1C3-BBa_B0032-cspD

pSB1C3-34-AOX1

pSB1C3-I13453-34-AOX1

pSB1C3-I13453-pSB1C3-BBa_B0032_cbtA

pSB1C3-oHybB-pSB1C3-E0240

pSB1C3_RNAG120_MlcRE-pSB1C3-BBa_I732018

pSB1C3-cspD

pSB1C3-accD

pSB1C3-ldhA

pSB1C3-ompT

pSB1C3-RNAG120

pSB1C3-NotMlcRE-HlyB-HlyD-TolC-OmpT

pSB1C3-pBAD-BjalT-OmpT-FLAG-HlyA

pSB1K3-NotMlcRE-HlyB-HlyD-TolC-OmpT

pSB1K3-pBAD-BjalT-OmpT-FLAG-HlyA

pSB1A3-NotMlcRE-HlyB-HlyD-TolC-OmpT

pSB1A3-pBAD-BjalT-OmpT-FLAG-HlyA

pSB1K3-RNAG120-MlcRE-pSB1C3-E0240

pSB1C3-RnaG120-Operator

pSB1K3-RNAG120-MlcRE-pSB1C3-BBa_B0034-ldhA

pSB1C3-RNAG120-MlcRE-pSB1C3-BBa_B0034-ldhA

pSB1C3-RNAG120-MlcRE-pSB1C3-E0240

pSB1C3-BBa_I13453-cbtA-cspD-mraZ

Vector

Vector

pSB1C3_RNAG120_MlcRE

pSB1C3-E0240

pSB1C3-oHybB

pSB1C3

pSB1C3-BBa_K206000

pSB1C3-BBa_B0032

pSB1C3

pSB1C3-I13453

pSB1C3-oHybB

pSB1C3_RNAG120_MlcRE

pSB1C3

pSB1K3

pSB1A3

pSB1K3

pSB1C3

pSB1K3

pSB1C3

pSB1C3-BBa_I13453

Tube

1

3

5

7

9

14

15

16

5

1

7

29

23

28

24

30

32

7

32

7

37

ρ

Gelex

18 ng/µL

33 ng/µL

8 ng/µL

50 ng/µL

Gelex

18 ng/µL

PCR Purification

19 ng/µL

PCR Purification

9 ng/µL

18 ng/µL

9 ng/µL

18 ng/µL

6 ng/µL

Länge

2697 bp

2200 bp

2070 bp

2200 bp

2697 bp

2070 bp

2200 bp

V

0.61 µL

4.25 µL

0.00 µL

0.29 µL

2.65 µL

1.01 µL

0.95 µL

0.98 µL

3.48 µL

0.17 µL

1.34 µL

1.49 µL

2.24 µL

1.39 µL

0.65 µL

0.70 µL

2.79 µL

Part 1

Part 1

pSB1C3-E0240

BBa_J45503

34-AOX1

mraz

32sulA

34sulA

cspD

34-AOX1

pSB1C3-BBa_B0032_cbtA

pSB1C3-E0240

pSB1C3-BBa_I732018

cspD

accD

ldhA

ompT

RNAG120

pGGA-NotMlcRE-HlyB-HlyD-TolC-OmpT

pGGA-pBAD-BjalT-OmpT-FLAG-HlyA

pGGA-NotMlcRE-HlyB-HlyD-TolC-OmpT

pGGA-pBAD-BjalT-OmpT-FLAG-HlyA

pGGA-NotMlcRE-HlyB-HlyD-TolC-OmpT

pGGA-pBAD-BjalT-OmpT-FLAG-HlyA

RNAG120-MlcRE

RnaG120-Operator

RNAG120-MlcRE

cbtA-cspD-mraZ

Tube

2

4

6

8

11

12

13

6

17

2

18

13

19

20

21

22

25

26

27

25

26

27

25

26

27

31

35

31

36

ρ

Gelex

10 ng/µL

Gelex

10 ng/µL

15 ng/µL

Gelex

2 ng/µL

Gelex

2 ng/µL

Gelex

2 ng/µL

30 ng/µL

15 ng/µL

30 ng/µL

15 ng/µL

Länge

878 bp

1200 bp

222 bp

1200 bp

394 bp

878 bp

252 bp

222 bp

915 bp

987 bp

954 bp

120 bp

6500 bp

602 bp

6500 bp

602 bp

6500 bp

602 bp

546 bp

300 bp

546 bp

1150 bp

V

10.00 µL

5.17 µL

8.16 µL

8.27 µL

8.22 µL

2.45 µL

32.71 µL

1.12 µL

3.94 µL

1.04 µL

1.12 µL

5.60 µL

Part 2

Part 2

pSB1C3-E0240

pSB1C3-BBa_B0034-ldhA

pSB1C3-E0240

Tube

33

34

33

ρ

Gelex

4 ng/µL

Gelex

Länge

878 bp

1000 bp

878 bp

V

15.31 µL

T4 Buffer

1.00 µL

2.00 µL

1.00 µL

2.00 µL

1.00 µL

2.00 µL

1.00 µL

4.00 µL

1.00 µL

4.00 µL

1.00 µL

4.00 µL

1.00 µL

3.00 µL

2.00 µL

1.00 µL

T4 Ligase

0.50 µL

H2O

8.50 µL

6.89 µL

5.63 µL

7.50 µL

8.21 µL

8.50 µL

0.68 µL

8.33 µL

8.28 µL

8.30 µL

2.58 µL

8.33 µL

2.79 µL

8.50 µL

1.45 µL

8.50 µL

2.79 µL

5.89 µL

2.31 µL

8.76 µL

0.50 µL

6.69 µL

0.11 µL

ad

10.00 µL

20.00 µL

10.00 µL

20.00 µL

10.00 µL

20.00 µL

10.00 µL

40.00 µL

10.00 µL

40.00 µL

10.00 µL

40.00 µL

10.00 µL

30.00 µL

20.00 µL

10.00 µL

Preparation of samples for DNA submission

Lotnummer	Well	Part	Konzentration	µl in well	noch genug vorhanden	Cryostock
181003NB03	1A	MlcRE	133.1	1.88	ja	aus 180908NF03
181009AK03	1B	oHybB	119	2.10	ja	180518BK06
181006LD01	1C	NOTMlcRE	53.364	4.68	ja	180712BK04
		RnaG120		#DIV/0!
180918AK12	1D	icsA	92	2.72	ja	180918AK05
181007NB05	1E	cspD	76.105	3.28	ja
180627LP03	1F	cbtA	65.5	3.82	ja	180627LP06
180914AW08	1G	mraZ	160	1.56	ja	180914LD03
180914AW01	1H	sulA	30	8.33	ja	180908NF04
181002NB09	2A	ldhA	65.203	3.83	ja	181002NB04
180918AK10	2B	accA	153	1.63	ja	180918AK03
180918AK09	2C	accB	65	3.85	ja	180918AK02
181003NB05	2D	accC	143.9	1.74	ja	180727DK04
		accD		#DIV/0!
		tesA		#DIV/0!
		LeuABCD		#DIV/0!
180726LP01	2E	kivD	107.39	2.33	ja	180727DK01
181009AK02	2F	ADH2	225	1.11	ja	180727DK03
181009AK01	2G	NnAOX1a	185	1.35	ja	180727DK02
180918AK14	2H	BjaIT-Linker-OmpTSite-FLAG	104	2.40	ja	180918AK07
181003NB10	3A	OmpT	137	1.82	ja
		NOT_MlcRE-BBa_B0030-accA-accD-BBa_B0032-accB-accC-BBa_B0031-tesA		#DIV/0!
		BBa_K206000-BBa_B0030-accA-accD-BBa_B0032-accB-accC-BBa_B0031-tesA		#DIV/0!
		NOT_MlcRE-ADH2-kivD-LeuABCD		#DIV/0!
		BBa_K206000-ADH2-kivD-LeuABCD		#DIV/0!
180918AK13	3B	oHybB-BBa_B0034-NnAOX1a	132	1.89	ja	180918AK06
180908NF05	3C	BBa_K206000-BBa_B0034-NnAOX1a	64.7	3.86	ja	180908NF01
		NOTMlcRE-BjaIT-OmpTSite-FLAG-HlyA		#DIV/0!
181006LD07	3D	BBa_K206000-BjaIT-OmpTSite-FLAG-HlyA	72.132	3.47	ja	181006LD11
		BBa_K206000-HlyB-HlyD-TolC-OmpT		#DIV/0!
180731NF03	3E	BBa_B0033-cbtA	174.8	1.43	ja	180531NF02
180914AW10	3F	BBa_B0032-cspD	77	3.25	ja	180531NF05
180731NF05	3G	BBa_B0033-cspD	110.1	2.27	ja	180531NF04
180703BK06	3H	BBa_B0032-mraZ	250	1.00	ja	180703LP06
180731NF02	4A	BBa_B0033-mraz	143.4	1.74	ja	180531NF01
180731NF06	4B	BBa_B0032-sulA	152.5	1.64	ja	180531NF06
180426NK28	4C	BBa_B0034-ldhA	85.514	2.92	ja	180518BK11
180703BK05	4D	BBa_B0032-accB	212	1.18	ja	180703LP05
180731NF01	4E	MlcRE-BBa_E0840	195.6	1.28	ja	180518BK02
180914AW07	4F	BBa_J45503-BBa_E0840	51	4.90	ja	180914LD02
180703BK02	4F	BBa_K206000-BBa_B0034-ldhA	167	1.50	ja	180703LP02
180703BK01	4G	BBa_K206000-BBa_B0032-ldhA	153	1.63	ja	180703LP01
181002NB08	4H	BBa_I13453-BBa_B0032-cbtA	157.52	1.59	ja	181002NB03
180914AW09	5A	BBa_K206000-BBa_B0032-sulA	104	2.40	ja	180914LD04
180817LD02	5B	pSB1C3-K206000-33cspD	132	1.89	ja	180826LP01
180426NK20	5C	pSB1C3-BBa_B0032-ldhA	63.0404	3.97	ja	180518BK10
180926NaF03	5D	pSB1C3-BBa_B0032-cbtA	93.011	2.69	ja	180531NF03
180731NF07	5E	psB1C3_BBa_B0034_sulA	167.9	1.49	ja	180518BK05
181003NB06	5G CAVE	pSB1C3-oHybB-BBa_E0240	167.6	1.49	ja
		pSB1C3-BBa_B0031-sulA		#DIV/0!		180518BK04
181006LD06	5F CAVE	pSB1C3_RNAG120_MlcRE-BBa_E0240 L1 #3	74	3.38	ja
181008DK04	5H	RNAG120-Operator	32.6	7.67
181010BK01	6A	BBa_I13453-BBa_B0034-cbtA-BBa_B0032-cspD-BBa_B0032-mraZ		#DIV/0!

Colony PCR

Colony PCRs of the transformations performed on October 8 were made.

Mastermix

Substance	1x (volume in µl)	20x (volume in µl)
10x DreamTaq Buffer	2	40
dNTPs 10mM	0.4	8
MgCl₂ 50mM	1.2	24
Fw-Primer VF2	0.6	12
Rv-Primer VR	0.6	12
Taq-Polymerase	0.1	2
ddH₂O ad 20µl	15.1	302

Colonies were picked with pipette-tips and transferred into 1 ml LB-Medium tubes after they were put in 20 µl aliquots of the mastermix of the following PCR reaction

PCR-Program (30 cycles)

Temperature (°C)	Time (s)
95	180
95	30
55	30
72	120
72	300

181009.png

Characterization of pSB1C3-BBa_K206000-BBa_B0034-cbtA-BBa_B0032-cspD-BBa_B0032-mraZ (growth inhibition module)

The ONCs set up on October 12 were used. 10 ml cultures were set up with a starting OD of 0.1

Measurements

Time (h)	OD BBa_I20270	OD DH5α	OD growth inhibiton module
0	0.01	0.01	0.01
1	0.025	0.032	0.021
2	0.084	0.149	0.058
3	0.380	0.728	0.260
4	1.09	1.65	0.81
5	1.58	1.75	1.16
6	1.88	2.24	1.67
7	2.33	2.57	2.12
8	3.27	3.13	2.58
9	2.98	3.20	2.18

Restriction, ligation and transformation

Restriction

	Tube	1	2
	Part	pSB1C3-E0240	RnaG120-MlcRE
	Lot#	181006LD02	IDT-syn
	ρ	53 ng/µL	100 ng/µL
	Use
	target m

Volumes	DNA	15.00 µL	2.50 µL
	NEBuffer 2.1	2.00 µL	1.00 µL
	EcoRI-HF	0.50 µL	0.50 µL
	XbaI	0.50 µL
	SpeI		0.50 µL
	PstI
	FastAP	0.50 µL
	H2O	1.50 µL	5.50 µL
	ad	20.00 µL	10.00 µL
	length Produckt

	ρ final	25 ng/µL	4 ng/µL

The probes were incubated at 37°C for 1h following another 20 min at 80 °C.

Ligation

Tube		1
Product		pSB1C3-E0240-RnaG120-MlcRE

Vector	Vector	pSB1C3-E0240
	Tube	1
	ρ	25 ng/µL
	Länge	2800 bp
	V	1.50 µL
Part 1	Part 1	RnaG120-MlcRE
	Tube	2
	ρ	4 ng/µL
	Länge	500 bp
	V	16.00 µL
Part 2	Part 2
	Tube
	ρ
	Länge
	V
T4 Buffer		2.00 µL
T4 Ligase		0.50 µL
H2O		0.00 µL
ad		20.00 µL

The probe was incubated at 37 °C for 1 h.

The product was transformed into DH5α.

150 µl Competent cell aliquots were thawed on ice for 15-30 min.
10 µl DNA were added and mixed
Cells were incubated for 35 min on ice
Heat-shock: 42°C for 45 sec, then put back on ice for 2-5 min
Pre-warm 850 µl LB-Medium
Add pre-warmed LB-Medium into the competent cells. 37°C 900 rpm 1 h
Cetrifuge 4500 rpm 2 min to collect cells.
Put cells on appropriate plates or medium
Incubation at 37°C overnight.

Colony PCR of pSB1C3-NOT-MlcRE-BBa_E0240

MasterMix

Substance	1x (volume in µl)	20x (volume in µl)
10x DreamTaq Buffer	2	40
dNTPs 10mM	0.4	8
MgCl₂ 50mM	0.6	12
Fw-Primer VF2	0.6	12
Rv-Primer VR	0.6	12
Taq-Polymerase	0.1	2
ddH₂O ad 20µl	15.7	314

Colonies were picked with pipette-tips and transferred into 1 ml LB-Medium tubes after they were put in 20 µl aliquots of the mastermix of the following PCR reaction

PCR-Program (30 cycles)

Temperature (°C)	Time (s)
95	180
95	30
54	30
72	120
72	300

The probes were applied to a 1% agarose gel

Colony one was characterized, as it showed fluorescence after incubation with glucose.

AOX1 characterization

Two 8 ml cultures were inoculated with the ONCs set up on October 14 to an OD₆₀₀ = 0.1 and incubated at room temperature; 120 rpm. At OD₆₀₀ = 0.2 the pSB1C3-BBa_K206000-BBa_B0034-AOX1 culture was induced with 10 µM arabinose.

Measurements

Time after induction [min]	Temperature DH5α	Temperature AOX1
0	23.8	23.8
60	24.4	24.5
120	24.5	24.5
150	25.5	25.5
180	25.8	25.8
210	25.8	25.7
240	25.4	25.3
270	25.3	25.2
300	25.1	25.0
330	25.0	24.9
390	23.7	23.7
430	24.2	24.2
470	24.3	24.3

Since the measurements didn’t show any significant differences the characterization needed to be repeated.

Plate	Colonies
pSB1C3_BBa_B0015	0
pSB1A2_BBa_E0040	~14
pSB1C3_BBa_E0840	4
pSB1C3_BBa_I13504	2
pSB1C3_BBa_I20270	12

Lot number	Part
180320MW01	Pre MlcRe suf
180320MW02	MlcRE without pre- and suffix
180320MW03	presulAsuf
180320MW04	sulA without pre- and suffix
180320MW05	BBa_B0031sulA
180320MW06	BBa_B0032sulA
180320MW07	BBa_B0034sulA
180320MW08	preoHybBsuf
180320MW09	oHybB without pre- and suffix

	M(tube)	m(Agarose)	Added buffer
Annealing:	0.9925 g	-	-
PCR:	0.9906 g	1.1417 g	151.1 µL

Lotnumber	Part
180208AK01	pSB1A2-BBa_E0040
180208AK02	pSB1C3-BBa_E0840
180208AK03	pSB1C3-BBa_I13504
180208AK04	pSB1C3-BBa_I20270

Substance	Volume (µL)
Vector pSB1C3-BBa_K206000	1.00
Part BBa_B0031-IdhA	4.77
T4 Buffer	2.00
T4 Ligase	0.50
H₂O	12.60

Substance	Volume (µL)
DNA	12.38
NF 2.1	2.00
EcoR/-HF	0.50
SpeI	0.50
H₂O	4.62

LOT-Number	Ligation
180425NK01	L3
180425NK02	L4
180425LD04	L5
180425LD05	L12
180425LD06	L16

LOT-Number	Concentration (ng/µL)
180426NK01	44.431
180426NK02	124.428
180426NK03	38.912
180426NK04	69.166
180426NK20	63.040
180426NK28	85.514
180426NK30	80.416

Lot number cryostocks	Part	Lot number plate
180518BK02	pSB1C3-MlcRE-BBa_E0840	180522NiF01
180518BK07	pSB1C3-oHybB-BBa_E0840	180522NiF02
180518BK08	pSB1C3-BBa_K20600-BBa_B0032-sulA	180522NiF03
180518BK09	pSB1C3-BBa_K20600-BBa_B0034-sulA	180522NiF04
180518BK12	pSB1C3-BBa_K20600-BBa_B0031-sulA	180522NiF05

pSB1C3-BBa_B0033-mraZ	180531LP01
pSB1C3-BBa_B0033-cbtA	180531LP02
pSB1C3-BBa_B0032-cbtA	180531LP03
pSB1C3-BBa_B0033-cspD	180531LP04
pSB1C3-BBa_B0032-cspD	180531LP05
pSB1C3-BBa_B0032-sulA	180531LP06

DH5a/pSB1C3-BBa_B0033-mraZ	180531NF01
DH5a/pSB1C3-BBa_B0033-cbtA	180531NF02
DH5a/pSB1C3-BBa_B0032-cbtA	180531NF03
DH5a/pSB1C3-BBa_B0033-cspD	180531NF04
DH5a/pSB1C3-BBa_B0032-cspD	180531NF05
DH5a/pSB1C3-BBa_B0032-sulA	180531NF06

Part	Well
BBa_R0040	2D
BBa_I20270	2B
BBa_J364000	2F
BBa_J364001	2H
BBa_J364002	2J
BBa_J364007	2L
BBa_J364008	2N
BBa_J364009	2P

Part	Lot-No.
pSB1C3-BBa_B0032-ldhA	180627LP01
pSB1C3-BBa_E0840	180627LP02
pSB1C3-cbtA	180627LP03

Vektor	psB1C3 (180628NB01)	RNA G120 Operator (180709NB01)
ρ	96.6 ng/µL	144.037 ng/µL
VDNA	1.04 µL	0.69 µL
NEB Buffer	1 µL	1 µL
EcoR1	0.5 µL	0.5 µL
Pst1	0.5 µL	0.5 µL
Fast AP	0.5 µL	-
H₂O_dest	6.46 µL	7.31 µL

psB1C3	3.05 µL
RNA G120 Operator	6.95 µL
T4-Buffer	1 µL
T4-Ligase	0.5 µL
H₂O_dest	-
Total	11.5 µL

Tube	Part	Size [bp]
1-5	ADH2	~1000
6-10	Accc	~1000
11-14	KivD	~1600
15-19	OmpA-Aox1a	~1200

Lotnumber	Part
180807BK01	3MB
180807BK02	Excretion
180807BK03	Myristic Acid
180807BK04	Growth Inhibition

Tube	Part
15-20	3MB
21-26	Excretion
27-32	Myristic Acid
33-38	Growth Inhibition

Plate		Kit	Lotnumber
Plate 3	8E	Kit2018	180822NiF01
Plate 6	19P	Kit2018	180822NiF02
Plate 6	20K	Kit2018	180822NiF03
Plate 6	10F	Kit2018	180822NiF04

Seq. ID	Lotnumber	Part	DNA-concentration [ng/µL]	length [bp]	Part length [bp]	Primer
98ED21	180817LD09	pSB1C3-pBAD-34cbta-34cspD-34-mraz-34sulA	191	1896	1136	GGA_cPCR_fw
98ED22	180817LD09	pSB1C3-pBAD-34cbta-34cspD-34-mraz-34sulA	191	1896	1136	GGA_cPCR_r
98ED23	180817LD03	pSB1C3-33killR	66	1896	250	Vf2

Part with the old lotnumber	New lotnumber
180826LP01	180817LD02
180826LP02	180817LD09
180826LP03	180817LD10
180826LP04	180817LD11
180826LP05	180817LD12
180826LP06	180817LD14

Tube	Part
1	pSB1C3-BBa_K20600-32-sulA
2-6	pSB1C3-B0840-oHybB
7	pSB1C3-cspD

Part	Lot number	DNA-concentration (ng/µL)
pSB1C3-I13500	180919AK01	151.75

Team:Hamburg/Notebook

Preparation of Ca/glycerol buffer and LB media

Preparation of agar plates

Overnight culture of DH5a

Preparation of competent cells

Test transformation

Resuspension of required plasmids

Transformation

Supplement to the transformation

Transformation

Examining the transformation

Transformation

Minipreparation of overnight cultures from 180206

Preparation of the overnight cultures for growing competent cells

Cryostocks of the overnight cultures from 180206

Preparation of competent cells

Transformation

Test transformation with cells from 180209 and by team Ignatova

Transformation with BBa_K206000 and BBa_B0015

Preparation of two overnight cultures (ONCs)

PCR for amplification of parts by E.coli

Minipreparation of BBa_K206000 and BBa_B0015

Nanodrop measurement

Restriction

Agarose gel electrophoresis

PCR for amplification of parts by E.coli

Restriction

Agarose gel electrophoresis of PCR products

Minipreparation

Nanodrop measurement

Retaking the PCR from 180312

Restriction

Gel extraction

Restriction

Ligation

Retaking the PCR from 180312

Setting up of Minipreps of the ONC prepared on the 21st of March

Restriction and ligation

Transformation of the ligations perfomed on the 21st of March

Colony PCR of the transformation performed on the 22nd of March

Repetition of the colony PCR performed on the 29th of March

PCR of multiple parts.

Restriction of pSB1C3 and BBa_K206000

Miniprep of a colony PCR

Repetition PCR ldHA/Annealing

Gelelectrophoresis:

Restriction of BBa_B0032_sulA and oHybB (Pre + Suffix)

Ligation of psB1c3_BBa-B0032_sulA (L12) and psB1C3_oHybB (L16)

PCR of BBa_B0031SulA, BBa_1718018 (Annealing)

Gelelektrophorese

Repetition of the PCR of BBa_I718018

Restriction of BBa_B0031_sulA mit XbaI and PstI

Ligation of pSB1C3-IdhA, pSB1C3-BBa_0031-IdhA, pSB1C3-BBa_B0032-IdhA and pSB1C3-BBa_B0034-IdhA

Repetition of the PCR of MlcRE with Pre and Suffix

Ligation of pSB1C3-BBa_K206000-BBa_B0031-SulA

Preparation of media

Gel electrophoresis of MlcRE with Pre and Suffix and BBa_I718018

Gelextraction of MlcRE with pre-and suffix

Restriction of MlcRE with pre-and suffix

Ligation of pSB1C3-MIcRE-BBa_B0031-IdhA, pSB1C3-MlcRE-BBa_B0032-idhA, pSB1C3-MlcRE-BBa-B0034-IdhA

Preparation of agar-Plates

Transformations of L3-L9, L24 preparation of agar-plates

Repetition of Transformations L3-L9, L24

Repetition of the PCR of MlcRE with Pre and Suffix

PCR purification using Gene Jet PCR Purification Kit (eluted with ddH2O)

Gel electrophoresis of MlcRE (PCR Product)

Repetition of Transformations L3-L9, L24, and Transformation of L12 and L16

Repetition of Ligations L12, L16 and L5

Transformations of L3 (pSB1C3-MlcRE-B0031-IdhA) and L4 (pSB1c3-MlcRE-BBa-B0032-IdhA), L5, L12, and L16

Colony PCR of L 3, L4, L6-L9, L24

Culture pSB1c3 for Minipreps was prepared

Overnight cultures of PCR-Products were prepared

Minipreps of 180426NK01, 02, 03, 04, 20, 28, 30

Set up of Midiprep from pSB1C3-BBa_I20270

Set up of Midiprep from pSB1C3-BBa_I20270

Resuspension of kit-DNA

Transformation of the kit-DNA into DH5α

Set up of ONCs

Set up of chloramphenicol-stocks

ONCs from the performed transformations of the 8th of March

Setting up of Minipreps of the ONC prepared on the 21^st of March

Transformation of the ligations perfomed on the 21^st of March

Colony PCR of the transformation performed on the 22^nd of March

Repetition of the colony PCR performed on the 29^th of March

PCR purification using Gene Jet PCR Purification Kit (eluted with ddH₂O)

ONCs from the performed transformations of the 8^th of March

ONCs from the performed transformations of the 8^th of March

Setting up Minipreps of the ONCs performed on the 13^th of March

cPCR of the transformation of the 19^th June

Repetition of the restriction and the ligation of the 14^th June

Old Lot number	New Lot number	Part	DNA-concentration (ng/µL)
180727DK02	180920OM01	pSB1C3-ompA-Aox1	513.055
180917AK02	180920OM02	pGGA-NOTMlcRE-HlyB-HlyD-TolC-OmpT	770.954

Lot number	Part
180803NB01	BjalT
180908NF01	AOX1
Plate Retrafo	BBa_I13500

Part	OD
DH5α	5.36
ldhA + Arabinose	4.784
ldhA without Arabinose	5.776

Clones	Part
1-8	pSB1C3-BBa_K20600-BBa_0034-sulA
9-16	pSB1C3-MlcRE (from 180518BK01)
17-19	pSB1C3-I13453-BBa_0034-AOX1

DH5α-control			Marker	ldhA with Arabinose			ldhA without Arabinose
2 µL	10 µL	20 µL	5 µL	2 µL	10 µL	20 µL	2 µL	10 µL	20 µL

	separating gel (15%)	stacking gel
Acrylamid	6.75 mL	750 µL
Separating gel buffer	4.5 mL	-
Stacking gel buffer	-	1.7 mL
H₂O_dest	6.75 mL	4.25 mL
10% APS	60 µL	20 µL
TEMED	13.5 µL	12 µL

Substance	1x volume (µl)
DNA-Template (pGGA)	10
BsaI-HF	0.5
10x CutSmart buffer	2
ddH₂O	7.5

Culture	Induction	Start-OD	OD after 6 h
DH5α (control)	-	0.076	5.22
ldhA (not induced)	-	0.036	8.02
ldhA (induced)	0.248ml arabinose	0.026	5.06
BjaIT (not induced)	-	0.182	2.4
BjaIT (induced)	0.224 ml arabinose	0.110	3.52

Substance	Volume
25 mM Tris	0.151g
150 mM NaCl	0.435g
0.5% Sodium desoxycholat	0.25g
1% NP-40 Alternative	500µl
ddH₂O	ad 50ml

	Stacking gel	Separate gel
Stacking gel buffer	0.675ml	-
Separate gel buffer	-	2.6ml
ddH₂O	3.425ml	3.2ml
Acrylamid	0.875ml	4.1ml
APS	30µl	60µl
TEMED	15µl	30µl