Team:SCUT-FSE-CHINA/Construction
Construction
N&P Pathways
The recombinant plasmid pETDuet-for-ptx was constructed by inserting the formamidase (FOR) gene amplified from the genome of Paenibacillus pasadenensis. CS0611 and the phosphite dehydrogenase (PTDH) gene from Klebsiella pneumonia. OU7 into two multiple cloning sites of pETDuet-1, respectively. The recombinant plasmid was then transfected into the hosts and we got E. coli DH5α (pETDuet-for-ptx) and E. coli BL21(DE3), successively.
① Using PCR, formamidase gene (for) was amplified from the genome of Paenibacillus pasadenensis. CS0611. pETDuet-for was constructed with for ligated into the first MCS in the pETDuet-1 vector, which was digested with BamHI/ EcoRI. Recombinant DH5α (pETDuet-for) was obtained after transfection.
② To find the phosphite dehydrogenase gene, we cultivated and screened some strains, collected from 7 sites, on a medium with phosphite as the sole phosphorus source and got the Klebsiella pneumonia strain with the ability to utilize phosphite.
Click here to see more details about this process.
③ Phosphite dehydrogenase gene (ptx) was similarly amplified from the genome of Klebsiella pneumonia. OU7, followed by ligation to the second MCS in pETDuet-for, which was digested with Bg1II/ KpnI. Since then, pETDuet-for-ptx has been constructed successfully. Recombinant DH5α(pETDuet-for-ptx) was obtained after transfection.
④ After completion of the circuit, we transfected our recombinant vector into E. coli expressing strain, which turned out to be the BL21(DE3)(pETDuet-for-ptx).
Next, BL21(DE3) (pETDuet-for-ptx) was inoculated and cultivated in LB medium, followed by induced expression with IPTG. Finally, the bacterial cells were collected and continually cultured in MOPS medium containing formamide and phosphite.
In the above demonstrated construction way, the formamidase (FOR) gene and the phosphite dehydrogenase (PTDH) gene were constructed in a co-expression vector, with dual promoters, which was named pETDuet-for-ptx.
Meanwhile, we were wondering if the modified metabolic pathways could be more efficient in other construction ways. So, these two genes were also constructed in a way of fusion protein in the pGEX-2T vector with GST tag, resulting in a recombinant vector named pETDuet-for-ptx.
CRIPSPR/Cas9
① The first N20 we planned to employ was required for synthesizing the DNA packaging protein of T7 phage. (It has been submitted to the registry in part BBa_K2325003). This part can be considered as an improvement of the part submitted by UBC2013 ( part BBa_K1129012), as they contain same N20 sequence but different sgRNA sequence. We fused crRNA and tracrRNA as a single synthetic guide RNA (sgRNA), while they separated the spacer and tracrRNA in their construct.
The way we constructed the sgRNA obviated the need for processing the transcribed CRISPR array (pre-crRNA) into individual crRNA components, helping to promote the function of resistance against T7 phage.
② For single-gene editing, after designing the N20 sequence, pTarget could thus be cloned simply by changing the N20 sequence of the sgRNA when different genomic loci are being targeted, which could be done by inverse PCR with mutations incorporated into the primers, resulting in the pTarget-N20. (The composite part has been submitted to the registry in part BBa_K2325104)
③ The E. coli strain BL21(DE3) was used as the host when pCas was also cotransformed with pTarget-N20. The recombinant strain BL21(DE3) (pCas+pTarget-N20) was obtained after cultivation on LB media with kanamycin and spectinomycin.
