Difference between revisions of "Part:BBa K4468000"
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<partinfo>BBa_K4468000 parameters</partinfo>
 
<partinfo>BBa_K4468000 parameters</partinfo>
 
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−
<h1>Usage and Biology</h1>
+
<h1>'''Usage and Biology'''</h1>
 
PmrC is a promoter that can bind to phosphorylated PmrA and initiate expression.
 
PmrC is a promoter that can bind to phosphorylated PmrA and initiate expression.
 
The PmrCAB of Salmonella is a system that can be stimulated and induced by exogenous metal ions. The main functions are executed by three parts, the transmembrane protein PmrB, the intracellular protein PmrA, and the promoter PmrC. In wild-type Salmonella, it can adsorbs extracellular Fe3+, and phosphorylates protein PmrA. Then the phosphorylated PmrA will bind on the promoter PmrC to initiate expression.
 
The PmrCAB of Salmonella is a system that can be stimulated and induced by exogenous metal ions. The main functions are executed by three parts, the transmembrane protein PmrB, the intracellular protein PmrA, and the promoter PmrC. In wild-type Salmonella, it can adsorbs extracellular Fe3+, and phosphorylates protein PmrA. Then the phosphorylated PmrA will bind on the promoter PmrC to initiate expression.
−
<h1>Molecular cloning</h1>
+
<h1>'''Molecular cloning'''</h1>
 
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.
 
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.
 
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.
 
Finally we transformed our recombinant plasmids into E. coli BL21(DE3) competent cells. Correct as checked by colony PCR.
 
[[File:HUST-China-00-1.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-00-1.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.]]
−
<h1>SDS-PAGE</h1>
+
<h1>'''SDS-PAGE'''</h1>
 
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.
 
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.
 
[[File:HUST-China-00-2.png|400px|thumb|center|Fig.2  SDS-PAGE result of membrane protein oprf-Sitag-LanM(PmrCAB) induced by different lanthanides.<br><br>After induction using different lanthanide ions, we obtained several strains that successfully expressed the oprf-Sitag-LanM(PmrCAB). All their membrane proteins were detected by SDS-PAGE. The band of oprf-Sitag-LanM(PmrCAB) is about 65kDa, identical to the theoretical length of 62.82kDa and still within explainable and acceptable range of glycosylation or phosphorylation modification. Oprf-Sitag-LanM(PmrCAB) could be confirmed as successfully expressed. Besides, following elution result also could verify it.]]
 
[[File:HUST-China-00-2.png|400px|thumb|center|Fig.2  SDS-PAGE result of membrane protein oprf-Sitag-LanM(PmrCAB) induced by different lanthanides.<br><br>After induction using different lanthanide ions, we obtained several strains that successfully expressed the oprf-Sitag-LanM(PmrCAB). All their membrane proteins were detected by SDS-PAGE. The band of oprf-Sitag-LanM(PmrCAB) is about 65kDa, identical to the theoretical length of 62.82kDa and still within explainable and acceptable range of glycosylation or phosphorylation modification. Oprf-Sitag-LanM(PmrCAB) could be confirmed as successfully expressed. Besides, following elution result also could verify it.]]
 
[[File:HUST-China-00-3.png|400px|thumb|center|Fig.3  SDS-PAGE result of membrane protein oprf-Sitag-FP(PmrCAB) induced by different lanthanides.<br><br>After induction using different lanthanide ions, we obtained several strains that successfully expressed the oprf-Sitag-FP(PmrCAB). All their membrane proteins were detected by SDS-PAGE. The band of oprf-Sitag-FP(PmrCAB) 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(PmrCAB) could be confirmed as successfully expressed. Besides, following elution result also could verify it.]]
 
[[File:HUST-China-00-3.png|400px|thumb|center|Fig.3  SDS-PAGE result of membrane protein oprf-Sitag-FP(PmrCAB) induced by different lanthanides.<br><br>After induction using different lanthanide ions, we obtained several strains that successfully expressed the oprf-Sitag-FP(PmrCAB). All their membrane proteins were detected by SDS-PAGE. The band of oprf-Sitag-FP(PmrCAB) 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(PmrCAB) could be confirmed as successfully expressed. Besides, following elution result also could verify it.]]

Revision as of 09:29, 30 September 2022


PmrC


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
    COMPATIBLE WITH RFC[25]
  • 1000
    COMPATIBLE WITH RFC[1000]


Usage and Biology

PmrC is a promoter that can bind to phosphorylated PmrA and initiate expression. The PmrCAB of Salmonella is a system that can be stimulated and induced by exogenous metal ions. The main functions are executed by three parts, the transmembrane protein PmrB, the intracellular protein PmrA, and the promoter PmrC. In wild-type Salmonella, it can adsorbs extracellular Fe3+, and phosphorylates protein PmrA. Then the phosphorylated PmrA will bind on the promoter PmrC 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.

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.2 SDS-PAGE result of membrane protein oprf-Sitag-LanM(PmrCAB) induced by different lanthanides.

After induction using different lanthanide ions, we obtained several strains that successfully expressed the oprf-Sitag-LanM(PmrCAB). All their membrane proteins were detected by SDS-PAGE. The band of oprf-Sitag-LanM(PmrCAB) is about 65kDa, identical to the theoretical length of 62.82kDa and still within explainable and acceptable range of glycosylation or phosphorylation modification. Oprf-Sitag-LanM(PmrCAB) could be confirmed as successfully expressed. Besides, following elution result also could verify it.
Fig.3 SDS-PAGE result of membrane protein oprf-Sitag-FP(PmrCAB) induced by different lanthanides.

After induction using different lanthanide ions, we obtained several strains that successfully expressed the oprf-Sitag-FP(PmrCAB). All their membrane proteins were detected by SDS-PAGE. The band of oprf-Sitag-FP(PmrCAB) 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(PmrCAB) could be confirmed as successfully expressed. Besides, following elution result also could verify it.