Part:BBa_K4468005

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 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

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

Fig.1 SDS-PAGE result of GolS after purification of total protein extraction product through Nickel-affinity chromatography column

The target protein located around 20kDa, similar the theoretical 17.51kDa. GolS could be confirmed as successfully expressed.

CUG-China 2024--Contribution

GolS is a gold-binding MerR family transcription factor found in Samonella sp., which enables the specific binding of Au[III] and triggers the transcription of downstream genes through the promoter P_gol. It was first registered in 2020.

Fig 3. The gene circuit of expression vector “pYDT-golS-Pgol-gfp”

In order to quantify the specific to Au[III], we designed the expression vector “pYDT-golS-P_gol-gfp” in E. coli BL21 to characterize its function.

Fig 4. (A) Plot of normalized fluorescence intensity of engineering bacteria E.coli BL21 in different metal ions. (B) Plot of normalized fluorescence intensity of engineered bacteria E.coli BL21 in different metal ions at 12 hours.

In the experiment, we added different metal salt solutions, HAuCl₄, NiCl₂, CdCl₂, CuSO₄, ZnCl₂, to the culture system of the engineered bacterium E.coli BL21, and the ion concentrations of metal ions Au³⁺, Ni²⁺, Cd²⁺, Cu²⁺ and Zn²⁺ were all 20 μM. By detecting the normalized fluorescence intensity of the engineered bacteria in different ion cultures. We found that the fluorescence intensity of 20 μM Au³⁺ culture was significantly higher than the fluorescence intensity produced when other metal ions were cultured. This indicates that the gene line golS-P_gol-gfp can respond specifically to Au[III].