Part:BBa_K4515012
Pcrt-crt-terohbd-Pthl-thl-opt
Pcrt-crt-terohbd-Pthl-thl-opt
Profile
Name: Pcrt-crt-ter-hbd-Pthl-thl-opt
Base Pairs: 4570 bp
Origin: Clostridium acetylbutyrate ATCC824 genomic DNA
Properties: A N-butanol metabolism pathway platform
Usage and Biology
This composite part is composed of the Pcrt promoter (BBa_K4515009), codon-optimized crt gene (BBa_K4515006), hbd gene (BBa_K4515005), ter gene (BBa_K4515007), Pthl promoter (BBa_K4515008), and thl gene (BBa_K4515004) [1-6], and was constructed into the pIB184 vector backbone to construct the pLY15-opt plasmid (Figure 1).
Experimental approach
1. PCR amplification of Pcrt-crt-ter-hbd-Pthl-thl-opt.
We designed the fused DNA fragment Pcrt-crt-ter-hbd-Pthl-thl-opt into ApaI and BglII sites of the pIB184 vector. In order to build our plasmids, we firstly amplified the gene fragments from the Clostridium acetylbutyrate ATCC824 genomic DNA (Figure 2), double-enzyme digestion, and ligase to pIB184 carrier.
In Figure 2, a clear and single DNA band for each gene was shown, indicating that the genes were successfully amplified by PCR.
Then, the PCR products were purified by gel extraction, four gene fragments were ligated to the pIB184 carrier by the one-step cloning method. Finally, recombinant plasmid pLY15-opt containing the Pcrt-crt-ter-hbd-Pthl-thl-opt DNA fragment was transformed into E. coli DH5α competent cells and coat on LB plate containing Erythromycin and incubated.
2. Transform plasmid pLY15-opt into L. Brevis ATCC367.
L. Brevis was cultured on a solid MRS plate, then a single colony was taken and cultured overnight in the MRS medium (Figure 3A), and remained 100 μL was mixed with an equal volume of 50% glycerin and stored at -80℃. Then the constructed plasmid (containing 4 codon-optimized genes) was transformed into the L. Brevis ATCC367 by electroporation method and incubated at 37℃ for 24-48 hours. We selected 7 monoclonal colonies, 1 was positive control, 8 was negative control (without any gene fragment or vector), 9 was blank control without any template (only enzyme, without 4 gene fragments), and 4 failed (Figure 3B).
References
1.Hickman AB, Dyda F. The casposon-encoded Cas1 protein from Aciduliprofundum boonei is a DNA integrase that generates target site duplications. Nucleic Acids Res. 2015 Dec 15;43(22):10576-87. doi: 10.1093/nar/gkv1180. PMID: 26573596
2.Krupovic M, Shmakov S, Makarova KS, Forterre P, Koonin EV. Recent Mobility of Casposons, Self-Synthesizing Transposons at the Origin of the CRISPR-Cas Immunity. Genome Biol Evol. 2016 Jan 13;8(2):375-86. doi:10.1093/gbe/evw006. PMID: 26764427; PMCID: PMC4779613.
3.Béguin P, Charpin N, Koonin EV, Forterre P, Krupovic M. Casposon integration shows strong target site preference and recapitulates protospacer integration by CRISPR-Cas systems. Nucleic Acids Res. 2016 Dec 1;44(21):10367-10376. doi: 10.1093/nar/gkw821. PMID: 27655632; PMCID: PMC5137440.
4.Krupovic M, Béguin P, Koonin EV. Casposons: mobile genetic elements that gave rise to the CRISPR-Cas adaptation machinery. Curr Opin Microbiol. 2017 Aug; 38:36-43. doi: 10.1016/j.mib.2017.04.004. PMID: 28472712; PMCID: PMC5665730.
5.Béguin P, Chekli Y, Sezonov G, Forterre P, Krupovic M. Sequence motifs recognized by the casposon integrase of Aciduliprofundum boonei. Nucleic Acids Res. 2019 Jul 9;47(12):6386-6395.doi:10.1093/nar/gkz447.PMID:31114911; PMCID: PMC6614799.
6.Wang X, Yuan Q, Zhang W, Ji S, Lv Y, Ren K, Lu M, Xiao Y. Sequence specific integration by the family 1 casposase from Candidatus Nitrosopumilus koreensis AR1. Nucleic Acids Res. 2021 Sep 27;49(17):9938-9952. doi: 10.1093/nar/gkab725. PMID: 34428286; PMCID: PMC8464041.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal NgoMIV site found at 2075
Illegal AgeI site found at 1743 - 1000COMPATIBLE WITH RFC[1000]
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