Team:BUCT/Notebook
June
6.11 We read the literature on suicide system and realized that MazEF is a pair of very valuable TA systems that can be applied to our project.
6.17 We thought about how to select "variables" as the switch of the suicide system, and finally selected "changes in oxygen concentration", and found a suitable oxygen promoter-phyb on NCBI.
July
7.10 We checked the literature to find the source of the target gene.
7.16 We obtained the MazF from the HZAU-China team as the toxin gene.
7.19 We got the endogenous E.coli MazE and MazF from E.coli BW25113 through PCR.
7.21 We designed primers and started the construction of suicide plasmids.
August
8.11 We built 5-HTP and suicide plasmids, and got through PCR, Agarose gel electrophoresis , gel recycle, enzyme digestion, ligation, and chemical transfer for PCD, mazE/F, trpE/C/B, and P4H.
8.12 We verified the plasmid and found that there was a problem with the pSA plasmid, and carried out test-tube culture of bacteria containing pSA.
8.13 The lac-mazF-T1-phyb-mazE-T1 suicide plasmid was successfully constructed, and was successfully verified.
8.14 We extracted the plasmid of the pCS-lac-mazF bacterial solution and verified that it was successful. Half of the suicide parts have been constructed correctly. We also inoculated pSA empty plasmid and pSA-lac-TrpB-P4H-PCD bacterial solution.
8.15 We extracted the pSA-lac-TrpB-P4H-PCD plasmid, verified by restriction enzyme digestion, and used pSA to construct a three-gene plasmid.
8.16 We extracted the vgb (plasmid)+mazE, pSA+P4H+PCD+TrpB plasmid, verified by restriction enzyme digestion.
8.17 We got the suicide plasmid sequencing result, which is correct.
8.18 We successfully constructed the vgb-mazE plasmid and ligated it with lac-mazF.
8.20 We carry out PCD amplification on PCD, P4H, and TrpB, and carry out gel recycle.
8.21 The extracted plasmid was successfully verified, and homologous recombination bacteria were cultivated.
8.22 We used the homologous recombination method pCS-lac-TrpB-P4H-PCD to extract the plasmid, and the restriction enzyme digestion verification was successful.
8.23 We successfully prepared high-efficiency Nissle 1917 competent, homologous recombination of lac-FadD-FadL plasmid and pCS-lac-gadB(mut)-gdhA plasmid, and connected pSA-AnTrpC-TrpE and lac-TrpB-P4H-PCD.
8.24 We introduced the plasmid into the target bacteria Nissle 1917.
8.25 We cultivated engineering bacteria.
8.26 Extract the plasmid.
8.27 We performed PCR again on the lac-TrpB-P4H-PCD-T1 frame and saved the strain.
8.28 We used the plasmid extracted from the bacteria as a template, and used FadD primers to verify the successful construction of the plasmid.
8.29 We combined 5-HTP plasmid (pSA-AnTrpC-TrpE-lac-TrpB-P4H-PCD) and also combined GABA & FadD-Fad (pCS-lac-gadB(mut)-gdhA-lacFadD-FadL)
8.30 We revisef the suicide part verification plan, verifying that "death can be induced under aerobic conditions" failed, and prepared to rebuild.
September
9.1 We learnt how to use anaerobic workstation to create an anaerobic environment for the growth of engineering bacteria, which was beneficial to the characterization of suicide system.
9.2 We homologous recombined pSA-lac-TrpEG-AnTrpC and lac-TrpB-P4H-PCD.
9.3 We reconstructed 5-HTP plasmid construction-homologous recombination system.
9.5We used two methods to construct the general flow chart of 5HTP framed plasmid simultaneously.
9.7We constructed suicide parts plasmid Gibson.
9.8 We carried out the transformation and coating of plasmid.
9.9 We activated Nissle bacteria, and constructed 5HTP framed plasmid by restriction enzyme ligation.
9.10 We prepared Nissle bacterial liquid, and made GABA fermentation to prepare pCS-gadB(mut)-gdhA.
9.14 We cultured nissle 1917 that was successfully introduced into plasmid in glove box.
9.15 We verified PCs-gadb (mut)-gdha-lac-fadd-fadl, the electrotransformed double plasmid 5HTP.
9.16 We constructed GABA+fad- plasmid.
9.17 We verified the previous plasmid by enzyme digestion, but the verification failed, and 5-HTP was ready to electrotransfer double plasmids.
9.18 We constructed 5HTP fermentation-double plasmid, which was no longer closed, but directly double-transformed, and it produced 5HTP, which was successfully verified by enzyme digestion.
9.19 We successfully constructed GABA+fad- plasmid.
9.20 We introduced 5HTP fermentation-double plasmid to prepare competent cells.
9.21 We successfully carried out GABA+fad fermentation-electrotransfer coating.
9.22 We carried out 5-HTP fermentation.
9.23 We used colorimetry to detect GABA fermentation, and the result was not satisfactory.
9.24 We tested GABA fermentation.
9.25 After constantly exploring the reaction conditions, we found that Nissle 1917 needs to grow very slowly in anaerobic environment, and it takes 48-72 hours to see obvious colonies, thus confirming the success of suicide system.
BUCT--iGEM
BUCT--iGEM
