Difference between revisions of "Part:BBa K4468008"

 
Line 2: Line 2:
 
__NOTOC__
 
__NOTOC__
 
<partinfo>BBa_K4468008 short</partinfo>
 
<partinfo>BBa_K4468008 short</partinfo>
−
 
−
Description
 
−
This is a composite component for the absorption and recovery of lanthanides, especially for Tb3+. It consists of T7-PmrA-T7-PmrB(dLBT)-T7 Terminator-PmrC-oprf-Sitag-T7 Terminator. Induced by IPTG, it can express PmrA and PmrB protein. Besides, lanthanide ions in the external solution can initiate the expression of oprf-Sitag and let our protein bind on silica column with its engineered bacteria together.
 
  
  
Line 19: Line 16:
 
<partinfo>BBa_K4468008 parameters</partinfo>
 
<partinfo>BBa_K4468008 parameters</partinfo>
 
<!-- -->
 
<!-- -->
 +
 +
<h1>'''Description'''</h1>
 +
This is a composite component for the absorption and recovery of lanthanides, especially for Tb<sup>3+</sup>. It consists of T7-PmrA-T7-PmrB(dLBT)-T7 Terminator-PmrC-oprf-Sitag-T7 Terminator. Induced by IPTG, it can express PmrA and PmrB protein. Besides, lanthanide ions in the external solution can initiate the expression of oprf-Sitag and let our protein bind on silica column with its engineered bacteria together.
 +
 +
<h1>'''Usage and Biology'''</h1>
 +
<h2>'''PmrA'''</h2>
 +
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 Fe<sup>3+</sup>, and phosphorylates protein PmrA. Then the phosphorylated PmrA will bind on the promoter PmrC to initiate expression.<br>
 +
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.
 +
<h2>'''PmrB(dLBT)'''</h2>
 +
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 Fe<sup>3+</sup>, and phosphorylates protein PmrA. Then the phosphorylated PmrA will bind on the promoter PmrC to initiate expression.<br>
 +
The expression product of PmrB is a single pass transmembrane protein whose 34to 64 amino acids are located outside the membrane to adsorb Fe<sup>3+</sup>. Near the C-terminus is a protein kinase that can phosphorylate PmrA. dLBT can highly absorb Tb<sup>3+</sup>, the most representative element of lanthanides. Thanks to HUST-China 2017, we successfully obtained the dLBT with the best adsorption effect from 12 different LBT domains. In that case, we turned its Fe<sup>3+</sup> absorb domain into dLBT, thus achieving the function of specifically adsorbing Tb<sup>3+</sup>.
 +
<h2>'''PmrC'''</h2>
 +
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 Fe<sup>3+</sup>, and phosphorylates protein PmrA. Then the phosphorylated PmrA will bind on the promoter PmrC to initiate expression.<br>
 +
PmrC is a promoter that can bind to phosphorylated PmrA and initiate expression.
 +
<h2>'''Oprf-Sitag'''</h2>
 +
Oprf-Sitag is a protein composed of oprf and Sitag peptides, which is used to bind on silica column with its bacteria together. 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.
 +
 +
<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.<br>
 +
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.

Revision as of 11:04, 30 September 2022


T7-PmrA-T7-PmrB-T7 Terminator-PmrC-Oprf-Sitag-T7 Terminator


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
    INCOMPATIBLE WITH RFC[1000]
    Illegal SapI.rc site found at 749


Description

This is a composite component for the absorption and recovery of lanthanides, especially for Tb3+. It consists of T7-PmrA-T7-PmrB(dLBT)-T7 Terminator-PmrC-oprf-Sitag-T7 Terminator. Induced by IPTG, it can express PmrA and PmrB protein. Besides, lanthanide ions in the external solution can initiate the expression of oprf-Sitag and let our protein bind on silica column with its engineered bacteria together.

Usage and Biology

PmrA

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.

PmrB(dLBT)

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 PmrB is a single pass transmembrane protein whose 34to 64 amino acids are located outside the membrane to adsorb Fe3+. Near the C-terminus is a protein kinase that can phosphorylate PmrA. dLBT can highly absorb Tb3+, the most representative element of lanthanides. Thanks to HUST-China 2017, we successfully obtained the dLBT with the best adsorption effect from 12 different LBT domains. In that case, we turned its Fe3+ absorb domain into dLBT, thus achieving the function of specifically adsorbing Tb3+.

PmrC

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.
PmrC is a promoter that can bind to phosphorylated PmrA and initiate expression.

Oprf-Sitag

Oprf-Sitag is a protein composed of oprf and Sitag peptides, which is used to bind on silica column with its bacteria together. 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.

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.