Difference between revisions of "Part:BBa K4468001"
Zhichao Li (Talk | contribs) |
Zhichao Li (Talk | contribs) |
||
Line 24: | Line 24: | ||
<h1>'''Molecular cloning'''</h1> | <h1>'''Molecular cloning'''</h1> | ||
− | + | Using E. coli to extract our plasmids. 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 | + | |
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. | ||
[[File:HUST-China-01-1.png|400px|thumb|center|Fig.1 Plasmid construction and colony PCR results of reconstructed plasmid with PmrC and PgolB promoter<br><br>All the bands are identical to the theoretical lengths, which could demonstrate that these plasmid are correctly constructed and successfully transformed into E.coli, confirmed by sequencing.]] | [[File:HUST-China-01-1.png|400px|thumb|center|Fig.1 Plasmid construction and colony PCR results of reconstructed plasmid with PmrC and PgolB promoter<br><br>All the bands are identical to the theoretical lengths, which could demonstrate that these plasmid are correctly constructed and successfully transformed into E.coli, confirmed by sequencing.]] |
Revision as of 01:30, 3 October 2022
PgolB
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal NgoMIV site found at 1802
Illegal AgeI site found at 50
Illegal AgeI site found at 399
Illegal AgeI site found at 1337 - 1000COMPATIBLE 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 responsible for regulating the promoter PgolB. Upon recognizing and adsorbing extracellular Au3+, the expression of downstream genes after promoter PgolB will be activated.
PgolB is the promoter of the GolS system. The expression of genes after PgolB is strictly regulated by GolS. In the absence of Au3+, GolS inhibits PgolB to prevent downstream genes’ expression. Whereas when GolS adsorbs exogenous Au3+, the inhibition turns to cease, allowing PgolB to initiate expression.
Molecular cloning
Using E. coli to extract our plasmids. 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
After confirming through colony PCR and sequencing, we used the successfully transformed E. coli BL21 (DE3) for expression. We induced with IPTG and Tb3+ or IPTG and Cu2+ then followed by cell disruption to detect membrane proteins, as our fusion proteins would be expressed on the cell membrane.