Notebook

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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

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

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

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

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

Substance	Volume (µL)
Taq-Buffer	80
dNTPs (10 mM)	16
MgCl₂ (50 mM)	24
VF2 (10 µL)	24
VF (10 µL)	24
H₂O	632

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

Cryostock	Part	Plate
180518BK02	pSB1C3-MlcRE-BBa_E0840	180522NF01
180518BK07	pSB1C3-oHybB-BBa_E0840	180522NF02
180518BK08	pSB1C3-BBa_K206000-BBa_B0032-sulA	180522NF03
180518BK09	pSB1C3-BBa_K206000-BBa_B0034-sulA	180522NF04
180518BK12	pSB1C3-BBa_K206000-BBa_B0031-sulA	180522NF05

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

Time [min]	OD
55	0.068
120	0.21
138	0.362

chemical	Volume (µL)
10X Taq buffer	80
dNTP 10 mM	16
50 mM MgCl₂	24
Fw-Primer VF2	48
Rv-Primer VR	48
Taq DNA Polymerase (5 u/µL)	1
H₂O	583

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

Team:Hamburg/Notebook

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

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