Difference between revisions of "Part:BBa K4468002"

 
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<partinfo>BBa_K4468002 short</partinfo>
  
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===Usage and Biology===
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<span class='h3bb'>Sequence and Features</span>
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<partinfo>BBa_K4468002 SequenceAndFeatures</partinfo>
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===Functional Parameters===
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<partinfo>BBa_K4468002 parameters</partinfo>
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<h1>'''Usage and Biology'''</h1>
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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.<br>
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The expression product of PmrA is an intracellular protein which can be actived during phosphorylation by protein kinase at the C-terminus of PmrB. Phosphorylated PmrA is able to bind on the promoter PmrC and initiate its downstream genes’ expression.
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<h1>'''Molecular cloning'''</h1>
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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.<br>
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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.<br>
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Finally we transformed our recombinant plasmids into E. coli BL21(DE3) competent cells. Correct as checked by colony PCR
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[[File:HUST-China-02-1.png|400px|thumb|center|Fig.1  SDS-PAGE result of PmrA from composite component BBa_K4468010 and BBa_K4468010 after purification of total protein extraction product through Nickel-affinity chromatography column<br><br>The target protein located around 26-30kDa, similar the theoretical 26.87kDa. PmrA could be confirmed as successfully expressed. The concentration of E. coli total protein is so high that huge amount of impure protein is included during elution. But due to difference from impure or permeate bands, its consistency among several times of elution, this band could be verified as our target FMO.]]

Revision as of 10:11, 30 September 2022

PmrA


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

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 expression product of PmrA is an intracellular protein which can be actived during phosphorylation by protein kinase at the C-terminus of PmrB. Phosphorylated PmrA is able to bind on the promoter PmrC and initiate its downstream genes’ 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 SDS-PAGE result of PmrA from composite component BBa_K4468010 and BBa_K4468010 after purification of total protein extraction product through Nickel-affinity chromatography column

The target protein located around 26-30kDa, similar the theoretical 26.87kDa. PmrA could be confirmed as successfully expressed. The concentration of E. coli total protein is so high that huge amount of impure protein is included during elution. But due to difference from impure or permeate bands, its consistency among several times of elution, this band could be verified as our target FMO.