Difference between revisions of "Part:BBa K3838789"
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| − | === | + | ===Engineering Success=== |
1. The DNA level | 1. The DNA level | ||
After the engineered bacteria were transformed, the target plasmid after DH5α transformation was extracted and verified by DNA gel electrophoresis. For Nissle1917, we extracted the target plasmid and amplified the SOD expression element on the plasmid, which was verified by DNA gel electrophoresis. We also carried out enzyme digestion verification on the plasmid, adding restriction endonuclease MIuI and SacI for double enzyme digestion, and obtained bands of expected size, as shown in Figure 1. This further indicates that our plasmid transformation is successful. | After the engineered bacteria were transformed, the target plasmid after DH5α transformation was extracted and verified by DNA gel electrophoresis. For Nissle1917, we extracted the target plasmid and amplified the SOD expression element on the plasmid, which was verified by DNA gel electrophoresis. We also carried out enzyme digestion verification on the plasmid, adding restriction endonuclease MIuI and SacI for double enzyme digestion, and obtained bands of expected size, as shown in Figure 1. This further indicates that our plasmid transformation is successful. | ||
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[[File:T--SZU-China-BBa K3838789-Fabv3.png|400px|thumb|center|Figure 3 A. Untransformed DH5α coated in LB without triclosan B. Transformed DH5α coated in LB without triclosan C. Untransformed DH5α was coated on LB D. with 0.625μM/ mL triclosan. The transformed DH5α was coated on LB E. with 0.625μM/ mL triclosan. Untransformed DH5α was coated on LB F. The transformed DH5α was coated on LB supplemented with 1.25μM/ mL triclosan]] | [[File:T--SZU-China-BBa K3838789-Fabv3.png|400px|thumb|center|Figure 3 A. Untransformed DH5α coated in LB without triclosan B. Transformed DH5α coated in LB without triclosan C. Untransformed DH5α was coated on LB D. with 0.625μM/ mL triclosan. The transformed DH5α was coated on LB E. with 0.625μM/ mL triclosan. Untransformed DH5α was coated on LB F. The transformed DH5α was coated on LB supplemented with 1.25μM/ mL triclosan]] | ||
[[File:T--SZU-China-BBa K3838789-Fabv4.png|400px|thumb|center|Figure 4. The left figure shows the single colony transfer plasmid plasmid obtained by 0.625um/mL triclosan plate, and the right figure shows the single colony transfer plasmid obtained by 1.25um/mL triclosan plate. The target plasmid size was 5477bp.]] | [[File:T--SZU-China-BBa K3838789-Fabv4.png|400px|thumb|center|Figure 4. The left figure shows the single colony transfer plasmid plasmid obtained by 0.625um/mL triclosan plate, and the right figure shows the single colony transfer plasmid obtained by 1.25um/mL triclosan plate. The target plasmid size was 5477bp.]] | ||
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| + | ===Usage and Biology=== | ||
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<span class='h3bb'>Sequence and Features</span> | <span class='h3bb'>Sequence and Features</span> | ||
Revision as of 12:40, 8 October 2021
J-FabV
Antibiotic resistance genes and antibiotics are frequently used to maintain plasmid vectors in bacterial hosts such as Escherichia coli. Due to the risk of spread of antibiotic resistance, the regulatory authorities discourage the use of antibiotic resistance genes/antibiotics for the maintenance of plasmid vectors in certain biotechnology applications. Overexpression of E. coli endogenous fabI gene and subsequent selection on Triclosan has been proposed as a practical alternative to traditional antibiotic selection systems. Literature has improved that Vibrio cholera FabV, a functional homologue of E. coli FabI, can be used as a suitable marker for the selection and maintenance of plasmid vectors in E. coli. Triclosan is FDA-approved as a safe, broad-spectrum antibacterial. It is widely used in household chemicals such as soap and toothpaste, and has also been found in human breast milk and urine. By expressing resistance genes, we gave triclosan resistance to engineering bacteria and achieved screening effect.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12INCOMPATIBLE WITH RFC[12]Illegal NheI site found at 7
Illegal NheI site found at 30 - 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]