Difference between revisions of "Part:BBa K4407010"
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We amplified a plasmid pMTL-Pthl-BS2 offered by team NJTech_China to obtain a linearized pMTL-Pthl vector. Then, we obtained the fragment of dps gene from the genome of D. wulumuqiensis R12 by PCR. The dps gene fragment and linearized pMTL-Pthl vector were ligated by the Gibson assembly method. Then, we ran a colony PCR for the transformed bacterial colony (E .coli JM109), inoculated the positive bacterial colonies, and extracted the plasmids, which were then confirmed to be pMTL-Pthl-dps by gene sequencing. | We amplified a plasmid pMTL-Pthl-BS2 offered by team NJTech_China to obtain a linearized pMTL-Pthl vector. Then, we obtained the fragment of dps gene from the genome of D. wulumuqiensis R12 by PCR. The dps gene fragment and linearized pMTL-Pthl vector were ligated by the Gibson assembly method. Then, we ran a colony PCR for the transformed bacterial colony (E .coli JM109), inoculated the positive bacterial colonies, and extracted the plasmids, which were then confirmed to be pMTL-Pthl-dps by gene sequencing. | ||
===Conjugation into ''C. tyrobutyricum''=== | ===Conjugation into ''C. tyrobutyricum''=== | ||
| − | After the recombinant plasmid pMTL-Pthl-dps was conjugated into C. tyrobutyricum using E. coli CA434 as the donor strain. The expression of dps protein in the recombinant bacteria was confirmed by SDS-PAGE. | + | After the recombinant plasmid pMTL-Pthl-dps was conjugated into ''C. tyrobutyricum'' using E. coli CA434 as the donor strain. The expression of dps protein in the recombinant bacteria was confirmed by SDS-PAGE. |
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===Functional Parameters=== | ===Functional Parameters=== | ||
<partinfo>BBa_K4407010 parameters</partinfo> | <partinfo>BBa_K4407010 parameters</partinfo> | ||
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Revision as of 08:30, 8 October 2022
Pthl-dps，Expression of dps with Pthl promoter
This part is responsible for expressing the dps protein under the control of a Pthl promotor, a ribosome binding site (RBS) and a terminator. It is composed of BBa_K3443002 (Pthl), BBa_K103015 (RBS), BBa_K4407001 (dps), and BBa_K3585002 (terminator). dps protects the bacteria from oxidative damage. It protects DNA against oxidative damage by binding with Fe2+ to prevent it from reacting with H2O2 (Fenton’s reaction). In our project, we used this device to enhance the viability of C. tyrobutyricum in aerobic environment.
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
Plasmid construction
We amplified a plasmid pMTL-Pthl-BS2 offered by team NJTech_China to obtain a linearized pMTL-Pthl vector. Then, we obtained the fragment of dps gene from the genome of D. wulumuqiensis R12 by PCR. The dps gene fragment and linearized pMTL-Pthl vector were ligated by the Gibson assembly method. Then, we ran a colony PCR for the transformed bacterial colony (E .coli JM109), inoculated the positive bacterial colonies, and extracted the plasmids, which were then confirmed to be pMTL-Pthl-dps by gene sequencing.
Conjugation into C. tyrobutyricum
After the recombinant plasmid pMTL-Pthl-dps was conjugated into C. tyrobutyricum using E. coli CA434 as the donor strain. The expression of dps protein in the recombinant bacteria was confirmed by SDS-PAGE.