Difference between revisions of "Part:BBa K2933227"
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===Usage and Biology===
 
===Usage and Biology===
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This composite part is made up with five basic parts, the RBSa, the linker g and the GST tag, the cutting site of Prescission Protease and our target protein MYX-1. It encodes a protein which is Myxo fused with GST tag. The fusion protein is about 53.9 kD. In order to gain the highly purified target protein, we add GST tag in N-terminal of Myxo and combine the two parts with the cutting site of Prescission Protease. The fusion protein can be cut off at the cutting site by Prescission Protease. It is convenient for us to purify our target protein.<br>
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This composite part is made up with five basic parts, the RBSa, the linker g and the GST tag, the cutting site of Prescission Protease and our target protein MYX-1. It encodes a protein which is MYX-1 fused with GST tag. The fusion protein is about 53.9 kD. In order to gain the highly purified target protein, we add GST tag in N-terminal of MYX-1 and combine the two parts with the cutting site of Prescission Protease. The fusion protein can be cut off at the cutting site by Prescission Protease. It is convenient for us to purify our target protein.<br>
 
===Molecular cloning===
 
===Molecular cloning===
 
First, we used the vector pGEX-6p-1 to construct our expression plasmid. And then we converted the plasmid constructed to ''E. coli'' DH5α to expand the plasmid largely.<br>
 
First, we used the vector pGEX-6p-1 to construct our expression plasmid. And then we converted the plasmid constructed to ''E. coli'' DH5α to expand the plasmid largely.<br>

Revision as of 13:37, 23 September 2019


RBS a+Linker g+GST+Linker e+MYX-1

This part consists of RBS a, protein coding sequence(GST+Linker e+MYX-1), the RBS and the protein coding sequence can be connected by linker g. The biological module can be build into E.coli for protein expression. This part can be prefaced with promoters of different strengths and types to regulate expression function.

Sequence and Features


Assembly Compatibility:
  • 10
    INCOMPATIBLE WITH RFC[10]
    Illegal PstI site found at 806
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal PstI site found at 806
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    INCOMPATIBLE WITH RFC[23]
    Illegal PstI site found at 806
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal PstI site found at 806
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal SapI.rc site found at 113


Usage and Biology

This composite part is made up with five basic parts, the RBSa, the linker g and the GST tag, the cutting site of Prescission Protease and our target protein MYX-1. It encodes a protein which is MYX-1 fused with GST tag. The fusion protein is about 53.9 kD. In order to gain the highly purified target protein, we add GST tag in N-terminal of MYX-1 and combine the two parts with the cutting site of Prescission Protease. The fusion protein can be cut off at the cutting site by Prescission Protease. It is convenient for us to purify our target protein.

Molecular cloning

First, we used the vector pGEX-6p-1 to construct our expression plasmid. And then we converted the plasmid constructed to E. coli DH5α to expand the plasmid largely.

T--TJUSLS China--MYX-1 PCRmeiqie.png T--TJUSLS China--MYX-1 meiqie.png
Figure 1. Left: The PCR result of MYX-1. Right: The verification results by enzyme digestion.

After verification, it was determined that the construction is successful. We converted the plasmid to E. coli BL21(DE3) for expression and purification.