Difference between revisions of "Team:NKU China/Notebook"

Construction of targeting vector : the upstream and downstream of luxS gene were combined by over-lapping PCR and ligated into plasmid pKSU.

Transformed pKSU-ΔluxS into GR286, and selected out positive clones.

The transformants were cultured at 42^oC with chloramphenicol to select single-crossover clones.

The single-crossover strains were then cultured at LB medium and passaged every 12 hours for 4 generation.

Cultured the last generation at medium with 5-fluorouracil to select double-crossover clones. Regretfully, we didn't get the double-crossover clones.

We cultured transformants at 42^oC with chloramphenicol again and selected the single-crossover clones successfully.

The single-crossover strains were then cultured at LB medium and passaged every 12 hours for 4 generations.

Cultured the last generation at medium with 5-fluorouracil to select double-crossover clones. We finally got our aimed strain—GR286ΔluxS.

Cultured the GR286ΔluxS strain and made it competence for future use.

Cloned the lsrACDB gene from Bacillus thuringiensis and ligated it to T-vector.

Transformed the T-lsrACDB into DH5α and coated plate, and then selected positive clones by colony PCR.

After restriction enzyme digestion verification, we sent them to sequencing. Unfortunately, the sequencing result showed some mutations in cloning gene.

We repeated the process of gene cloning but there were still some mutations.

We finally decided to request the gene company to synthesize the lsrACDB gene.

We cloned a strong promoter C2 from former kit and cloned luxS gene from GR286.

Fuse the two segments together by fusion PCR, and ligated it into T-vector. Then, transformed the vector into DH5α.

Selected the positive clones by colony PCR.

We chose 4 positive strains to culture overnight and extracted the plasmid. After restriction enzyme digestion verification, we sent them to sequencing.

The sequencing result showed there's a correct strain. So we can use the strain for the following experiment. We obtained the correct plasmid T-C2-luxS from DH5α. Then we got the fragment C2-luxS by digestion and gel extraction.

Ligated the C2-luxS to linearized plasmid pWH1520, and transformed it into DH5α.

Extracted the plasmid pWH-C2-luxS from DH5α. To prevent the plasmid from DAM&DCM methylation, we transformed it into E.coli JM110.

Extracted the plasmid pWH-C2-luxS from JM110,and dealt with it by BamH Ⅰ methylase.

Transformed the plasmid into GR286 by electroporation.[Failed]

This week, we tried to use different voltages to transform the plasmid. Sadly, all of these tries got bad results.

We considered whether the luxS gene is a little toxic for GR286, and the bacteria tends to refuse the gene when we added a strong promoter in front of it. So, we planned to use induced expression to reconstruction our expression vector.

The plasmid pWH1520 contains the strong xylA promoter originating, and transcription from this promoter is xylose inducible. So, the gene of interest carries its own ribosome binding sequence (RBS) and translation initiation codon. Based on these points, we redesigned primers.

We cloned luxS gene from GR286 using our new primers.

Purified the luxS fragment by gel extraction, and ligated it into linearized pWH1520. Then, transformed the vector into DH5α.

Selected the positive clones by colony PCR.

We chose 4 positive strains to culture overnight and extracted the plasmid. After restriction enzyme digestion verification, we sent them to sequencing.

The sequencing result showed there's three correct strains. So we can choose a correct strain for the following experiment. We extracted the correct plasmid pWH-luxS from DH5α. To prevent the plasmid from DAM&DCM methylation, we transformed it into E.coli JM110.

Extracted the plasmid pWH -luxS from JM110,and dealt with it by BamH Ⅰ methylase.

Transformed the plasmid into GR286 by electroporation, and selected positive clones.

Our synthetic lsrACDB gene came back. We first used restriction-ligation method to ligate lsrACDB to plasmid pWH1520, but we failed to select positive after several tries.

Considering the lsrACDB gene is a large fragment (4500bp), we used ClonExpress technique cloning the gene again to improve the efficiency of ligation. We divided the lsrACDB sequence into two parts and cloned them separately. Then we ligated the two segments to the plasmid pWH1520 and transformed it into DH5α. After that, we used PCR to select the positive clones. However, we didn't get a good result.

Since we have constructed "supplier" and part of "consumer", we decided to measure the growth curve to explore the function of our "controller".