Difference between revisions of "Part:BBa K3838789"

 
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===Usage and Biology===
 
===Usage and Biology===
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1. The DNA level
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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 K3838798-Fabv1.png|400px|thumb|center|Figure 1:A. Gel electrophoresis of DNA transformed into EScherichia coli DH5α by plasmid PJSG (5939bp). B.After transformation, Nissle 1917 SOD expression element was amplified(target fragment size was 1890bp) c. The PJSG was double-digested with restriction endonuclease MIuI and SacI, and the expected band sizes were 1918bp and 4021bp]]
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2. Protein level
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The experimental group was DH5α with PJSG plasmid, and the control group was DH5α empty vector. As can be seen from Figure 2, protein expression in the experimental group was near 45kDa, which was consistent with our target protein band of 44kDa. It indicated that FabV protein was successfully expressed by the engineered bacteria.
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[[File:T--SZU-China-BBa K3838789-Fabv2.png|400px|thumb|center|Figure 2.sds-page of DH5α intracellular protein containing PJSG plasmid was performed. The untransformed DH5α was used as blank control group. The target protein size was 44kDa]]
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3. Functional characterization
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0.625μM/ mL triclosan concentration: there was no colony growth in the control group, but a few colonies grew in the experimental group. We selected a single colony and transferred it to the liquid before plasmid extraction for gel running and enzyme digestion verification. The target band was 5477bp, proving that the growing colony was indeed a transpromoter.
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DH5a:
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In order to find the effective working concentration of this screening system, LB plates were prepared at 0.625μM/ mL triclosan concentration、1.25μM/ mL triclosan concentration and without resistance, The DH5α bacterial solution at the early logarithmic growth stage was coated on the plate on the medium (the concentration of the two groups was controlled at about OD0.4).Place the plates at 37℃ in a constant temperature incubator for 18h, and obtain the results, as shown in Figure 3.
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Non-resistant group: both kinds of bacteria are overgrown on the plate;
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1.25μM/ mL triclosan concentration: There was no colony growth in the control group, but a few colonies grew in the experimental group. We selected a single colony and transferred it to the liquid, then plasmid was extracted for gel running and enzyme digestion verification, and the target band was 5477bp, proving that the growing colony was indeed a transpromoter.
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To further confirm, triclosan plates with 0.625um and 1.25um concentrations were re-selected for plasmid raising validation after the 4C plate was retained for 14 days, and the target bands were also obtained, as shown in Figure 4 In conclusion,0.625uM/ mL and 1.25uM/ mL triclosan concentrations can be used as screening conditions for non-antibiotic DH5α engineering bacteria.
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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]]
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[[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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<span class='h3bb'>Sequence and Features</span>
 
<span class='h3bb'>Sequence and Features</span>

Revision as of 12:36, 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


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal NheI site found at 7
    Illegal NheI site found at 30
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    COMPATIBLE WITH RFC[1000]