Part:BBa_K4468001

PgolB

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE 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
  
• 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 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

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.

Fig.1 Plasmid construction and colony PCR results of reconstructed plasmid with PmrC and PgolB promoter

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.

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.

Fig.3 SDS-PAGE result of membrane protein oprf-Sitag-FP(GolS).

The band of oprf-Sitag-FP(GolS) is about 70kDa, identical to the theoretical length of 68.03kDa and still within explainable and acceptable range of glycosylation or phosphorylation modification. Oprf-Sitag-FP(GolS) could be confirmed as successfully expressed. Besides, following elution result also could verify it.