Team:Goettingen/Notebook/reporterSystems September
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="">
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="">
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="">
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.