Difference between revisions of "Part:BBa K4468005"
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Using E. coli to extraction. Through designed primers, we have obtained different high copies linearized fragments from our plasmids by PCR. These fragments are then connected together by homologous recombination to form a complete plasmid. After transformed into E. coli, colony PCR was applied for confirmation. Then we go for extracting plasmids again.<br>
 
Using E. coli to extraction. Through designed primers, we have obtained different high copies linearized fragments from our plasmids by PCR. These fragments are then connected together by homologous recombination to form a complete plasmid. After transformed into E. coli, colony PCR was applied for confirmation. Then we go for extracting plasmids again.<br>
 
Finally we transformed our recombinant plasmids into E. coli BL21(DE3) competent cells. Correct as checked by colony PCR.
 
Finally we transformed our recombinant plasmids into E. coli BL21(DE3) competent cells. Correct as checked by colony PCR.
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Revision as of 10:37, 30 September 2022


GolS


Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BglII site found at 162
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    COMPATIBLE WITH RFC[1000]



Usage and Biology

The GolS of Salmonella is a transcriptional regulator comes from the gold specific family MerR. Its expressed protein GolS plays a role as an operon that responsibles for regulating the promoter PgolB. Upon recognizing and adsorbing extracellular Au3+, the expression of downstream genes after promoter PgolB will be activated.
GolS, which has a metal binding loop that can specifically binds Au3+ and regulates the expression of genes downstream of PgolB, is the most important part of the regulatory system. Literatures have proved that residues 108-120 of GolS, which are in this metal binding loop, play a decisive role for binding Au3+. It was found that if this part of the residues were changed to residues 108-120 of the cognate CueR protein, the new GolS protein not only has Au3+ adsorption ability, but also high Cu2+ adsorption. CueR and GolS are homologous transcription factors with high sequence similarity and similar metal binding loop. The difference is that CueR specifically binds Cu2+ rather than Au3+. Considering that the main application occasion of our project is mining wastewater, which tends to contain a large amount of copper element instead of gold element. So we chose the new gols, which replaced the residues of CueR, as our project protein and activate the expression of PgolB by Cu2+ induction.


Molecular cloning

First of all, we need to amplificated all the commercially synthesized plasmid to acquire enough amount for further study. After transformation, colony PCR is applied for confirmation. Then we go for plasmid extraction.
Using E. coli to extraction. Through designed primers, we have obtained different high copies linearized fragments from our plasmids by PCR. These fragments are then connected together by homologous recombination to form a complete plasmid. After transformed into E. coli, colony PCR was applied for confirmation. Then we go for extracting plasmids again.
Finally we transformed our recombinant plasmids into E. coli BL21(DE3) competent cells. Correct as checked by colony PCR.


SDS-PAGE