Team:Goettingen/Notebook/reporterSystems September

14.09.18

A PCR was performed to fuse the promotors PyciAB and PyciC to xylR.

Component Volume in [µL]
5x HF buffer 10
dNTPs (12.5 mM) 2
fwd Primer: iGEM2018_83 or iGEM2018_85 2
rev Primer: iGEM2018_90 2
B. subtilis SP1 chromosomal DNA 1
Phusion DNA polymerase 0.25
sterile H2O 32.75
Total 50
Primers Product
iGEM2018_83 + iGEM2018_90 PyciAB
iGEM2018_85 + iGEM2018_90 PyciC

The annealing temperature was set for 54°C and lasted for 1 minute. The elongatin settings were 2 min and 72°C.

   <img src="T--Goettingen--PCR_170918.png">

Lane 1 and 3:PyciC-xylR, lane 2 PyciAB-xylR

19.09.18

Compound Volume in [μL]
Fragment 20
BamHI 2
EcoRI 2
FD buffer 3
H2O 3
30 min at 37°C

The sample was purified with the DNA purification kit and eluted in 32 μL. Also, the DNA concentration was measured with the Nanodrop device.

Compound Volume in [μL]
pAC7 20
BamHI 3
FD buffer 3
H2O 4
45 min at 37°C

1.5 μL of the digested plasmid were put on an agarose gel next to a not digested plasmid sample.

   <img src="T--Goettingen--180918_restriktionsverdau.png">

Lane 1: undigested pAC7, lane 2: digested pAC7

Compound Volume in [μL]
pAC7 20
EcoRI 3
FD buffer 3
H2O 4
30 min at 37°C

The reaction was inactivated for 5 min at 80°C. Afterwards 1 μL fast AP was added and the reaction was then incubated for 10 min at 37°C. The sample was purified with the DNA purification kit and eluted in 32 μL water. The framgemts were ligated with pAC7 and a religation was performed along side the ligation reaction.

Compound Volume in [μL]
Vector (pAC7) 1
Insert (PyciAB/C-xylR) 2 (AB) 1 (C)
T4 buffer 2
T4 ligase 2
H2O 13 (AB) 14 (C)
at least 2 h at RT
Compound Volume in [μL]
Vector (pAC7) 1
T4 buffer 2
T4 ligase 2
H2O 15
at least 2 h at RT

Competent DH5α cells were transformed with the ligation samples and the cells were plated on LB Amp plates containing x-gal.

20.09.18

Inoculation of 15 ml LB over night cultures of three colonies each that showed a blue coulor.

21.09.18

A plasmid prep was performed and the DNA concentration was measured using the nanodrop.

Plasmid DNA concentration in [ng/µL]
PyciAB-xylR sample 1 250.1
PyciAB-xylR sample 2 317.0
PyciAB-xylR sample 3 269.4
PyciC-xylR sample 1 250.4
PyciC-xylR sample 2 245.2
PyciC-xylR sample 3 242.7

A test digest was performed.

Compound Volume in [µL]
BamHI 1
EcoRI 1
FD buffer 1
Plasmid 2
H2O 5
Incubate for 15 min at 37°C

The whole digestion sample was transferred to an agarosegel.

   <img src="T--Goettingen--200918.png">

Lane 1 to 3: PyciAB-xylR clone 1 to 3, lane 4 to 6 PyciC-xylR clone 1 to 3.

The colones 1 and 3 of PyciAB-xylR and clone 3 of :PyciC-xylR were send away for sequnecing. Clone 3 of :PyciAB-xylR came back positiv.

24.09.18

The strain BP270 was made competent. Next, the competent cells were transformed with pAC7 containing PyciAB-xylR isolated from clone 3. In a second reaction BP270 cells were transformed using only the empty pAC7 vector

24.09.18

6 colonies of each transformation were streaked out on agar plates to gain single colonies.

25.09.18

To test whether the correct integartion of the construct was successfull, the α amylase-activity was determined using starch agar plates. Here we determined that all picked clones of the transformation using only the empty plasmid were correct while only one of the 6 picked clones was correct for the transformation with the whole construct. The correct clone was used for further experiments.