Part:BBa_K4468012

T7- GolS- T7 Terminator- PgolB- Oprf-Sitag-LanM- T7 Terminator

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal NheI site found at 3082
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BglII site found at 191
  Illegal BamHI site found at 4012
  Illegal XhoI site found at 3424
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal NgoMIV site found at 2414
  Illegal AgeI site found at 662
  Illegal AgeI site found at 1011
  Illegal AgeI site found at 1949
  Illegal AgeI site found at 2866
  
• 1000
  COMPATIBLE WITH RFC[1000]

Description

This is a composite component for the absorption and recovery of rare earth elements, especially for lanthanides. It consists of T7-GolS- PgolB- oprf-Sitag-LanM- T7 Terminator. It can express GolS protein with IPTG induction. Besides, copper ions in the external solution can initiate the expression of oprf-Sitag-LanM protein that can achieve a large amount of lanthanide adsorption and attachment on the surface of silica column.

Usage and Biology

GolS

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.

PgolB

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.
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 inhibites PgolB to prevent downstream genes’ expression. Whereas when GolS adsorbs exogenous Au³⁺, the inhibition turns to cease, allowing PgolB to initiate expression.

Oprf-Sitag-LanM

Oprf-Sitag-LanM is a protein composed of oprf, Sitag and LanM peptides. It is the main element for adsorption and recovery of lanthanides. Oprf has a membrane-binding domain, which helps the protein binding on the cell membrane of our engineered bacteria. Sitag is a tag that can connect with silicon element. It allows us to easily fix our protein just using a silica column. LanM has great characteristics of efficient and specific absorbing lanthanides which can effectively absorb the free lanthanides in the environment. Through GS linker to combine them in a whole, we have created a new protein that can stick on our E.coli membrane and fix to silica column with its engineered bacteria together. When the mining wastewater flows through the column, the lanthanides can be effectively adsorbed, so as to achieve the purpose of rare earth element recovery.

Fig.1 The absorption principle of Sitag.

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.

Fig.2 Colony PCR result of Oprf-Sitag-LanM transformed E.coli

The band of Oprf-Sitag-LanM from colony PCR is about 1800bp, identical to the theoretical length of 1797bp estimated by the designed primer location, which could demonstrate that this target plasmid had successfully transformed into E.coli

Fig.3 Colony PCR result of our four pathways transformed E. coli

The band of PmrCAB-LanM from colony PCR is about 5000bp, identical to the theoretical length of 5156bp estimated by the designed primer location, the band of PmrCAB-FP from colony PCR is about 5000bp also identical to the theoretical length of 5399bp. Besides, the band of GolS-LanM from colony PCR is about 4900bp identical to the theoretical length of 5049bp and he band of GolS-LanM from colony PCR is about 5000bp identical to the theoretical length of 5196bp, which could demonstrate that the target plasmids had successfully transformed into E. coli.

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.

Fig.4 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.

Fig.5 SDS-PAGE result of membrane protein oprf-Sitag-LanM(GolS).

The band of oprf-Sitag-LanM(GolS) is about 60kDa, identical to the theoretical length of 62.82kDa and still within explainable and acceptable range of glycosylation or phosphorylation modification. Oprf-Sitag-LanM(GolS) could be confirmed as successfully expressed. Besides, following elution result also could verify it.

Fluorescence characterization

Fig.6 Expression of GFP in E. coli under microscope

We constructed a plasmid that GFP gene wass in downstream of promoter PgolB and transformed it into E. coli cells. After that we added IPTG induced its expression. Colonies after 24 hours of culture, of which we picked one and verified the expression of GFP. As can be seen, all the cells fluoresced, indicating the successful expression of GFP. The GolS pathway is working properly.

Silica absorbability

Fig.7 Validation of silica absorbability.

Picture A shows the silica observed under the electron microscope after mixing with our target strain for 5h. Picture B shows the untreated silica. Comparing A and B, the silica in Picture A binds a large number of protein molecules, indicating that our fusion protein can bind to silica successfully.

Fig.8 Silica adsorption curve. Red line: control group. Black line: experimental group.

Measuring the absorbance of medium solution and drawing the relation curve between OD and time. With the increase of silica adsorption time, the OD value of the solution has decreased obviously. It proved that the fusion protein has good absorbability indeed, which is consistent with the results obtained by electron microscopy.

Immunofluorescence labeling

Fig.9 Image of immunofluorescence labeling of the fusion protein from E. coli.

It is obvious to see that our target proteins are successfully expressed and immobilized on the cell membrane through the fluorescence on the bacteria cell.

Absorption of REE

Fig.10 REE absorbed from solution.

This is the REE absorption of Oprf-Sitag-LanM. The abscissa is 8 kinds of REE and the ordinate is the absorption of REE.

Fig.11 Absorption rate of Tb.

This is the REE absorption of Tb. The abscissa is 4 kinds of pathways: PmrL is short for BBa_K4468010. PmrFP is short for BBa_K4468011. GolSL is short for BBa_K4468012. GolSFP is short for BBa_K4468013. And the ordinate is the absorption of Tb. For our fusion proteins, the adsorption of Tb was great. The lowest adsorption rate is about 60% and the highest rate is more than 80%.