Difference between revisions of "Part:BBa K4885004"
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==Results== | ==Results== | ||
===(1)Plasmid construction and transformation=== | ===(1)Plasmid construction and transformation=== | ||
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We used the recombinant plasmid Pthl-adhE2 constructed by BBa_K4408008 as the template, Vad-f and Vad-r as the primers, and amplified to obtain the Vpthl-adhE2 vector (7985 bp). Using the genomic DNA of C. tyrobutyricum as a template, with primers Cat-f and Cat-r, we amplified a fragment of the butyryl-CoA/acetate CoA transferase (cat1) gene (1378 bp). The cat1 fragment and Vpthl-adhE2 linearized vector were ligated by Gibson assembly method. Colony PCR (1333 bp) was performed on the transformed colonies with primers cat-PF and Pb-PR. The positive colonies were transfected, plasmid was extracted, and the recombinant plasmid, pMTL-Pthl-adhE2-Pcat1-Cat1, was verified by gene sequencing. | We used the recombinant plasmid Pthl-adhE2 constructed by BBa_K4408008 as the template, Vad-f and Vad-r as the primers, and amplified to obtain the Vpthl-adhE2 vector (7985 bp). Using the genomic DNA of C. tyrobutyricum as a template, with primers Cat-f and Cat-r, we amplified a fragment of the butyryl-CoA/acetate CoA transferase (cat1) gene (1378 bp). The cat1 fragment and Vpthl-adhE2 linearized vector were ligated by Gibson assembly method. Colony PCR (1333 bp) was performed on the transformed colonies with primers cat-PF and Pb-PR. The positive colonies were transfected, plasmid was extracted, and the recombinant plasmid, pMTL-Pthl-adhE2-Pcat1-Cat1, was verified by gene sequencing. | ||
| + | ===(2)Construction of C. tyrobutyricum L319 with pMTL-Pthl-adhE2-cat1 === | ||
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Revision as of 06:45, 30 September 2023
Pthl-adhE2-Pcat1-Cat1
This part is responsible for the expression of adhE2 gene with Pthl promotor and Cat1 gene with Pcatl promotor. adhE2 gene is derived from Clostridium acetobutylicum. Cat1 gene is derived from Clostridium tyrobutyricum (C. tyrobutyricum) L319. It consists of Pthl sequence (BBa_K3443002), strong ribosomal binding site (RBS) sequence (BBa_K103015), adhE2 sequence (BBa_K1462060), terminator sequence (BBa_K3585002), Pcat1 sequence (BBa_K4119011), strong ribosomal binding site (RBS) sequence (BBa_K103015), Cat1 gene sequence (BBa_K4119013) and terminator sequence (BBa_K3585002). adhE2 gene encodes for the alcohol/aldehyde bifunctional dehydrogenase. Its role is to convert acyl-CoA to aldehyde then to alcohol in two reductive steps using NADH as cofactor. Cat1 gene codes the butyryl-CoA/acetate CoA transferase.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]
Results
(1)Plasmid construction and transformation
We used the recombinant plasmid Pthl-adhE2 constructed by BBa_K4408008 as the template, Vad-f and Vad-r as the primers, and amplified to obtain the Vpthl-adhE2 vector (7985 bp). Using the genomic DNA of C. tyrobutyricum as a template, with primers Cat-f and Cat-r, we amplified a fragment of the butyryl-CoA/acetate CoA transferase (cat1) gene (1378 bp). The cat1 fragment and Vpthl-adhE2 linearized vector were ligated by Gibson assembly method. Colony PCR (1333 bp) was performed on the transformed colonies with primers cat-PF and Pb-PR. The positive colonies were transfected, plasmid was extracted, and the recombinant plasmid, pMTL-Pthl-adhE2-Pcat1-Cat1, was verified by gene sequencing.