Difference between revisions of "Part:BBa K2933165"

 
Line 18: Line 18:
 
<partinfo>BBa_K2933165 parameters</partinfo>
 
<partinfo>BBa_K2933165 parameters</partinfo>
 
<!-- -->
 
<!-- -->
 +
===Usage and Biology===
 +
This composite part is made up with three basic parts, the GST tag, the cutting site of Prescission Protease and our target protein Myxo. 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>
 +
===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>
 +
<p style="text-align: center;">
 +
  [[File:T--TJUSLS China--MYX-1 PCRmeiqie.png|300px]]  [[File:T--TJUSLS China--MYX-1 meiqie.png|300px]]<br>
 +
'''Figure 1.'''  Left: The PCR result of MYX-1. Right: The verification results by enzyme digestion.<br>
 +
</p>
 +
After verification, it was determined that the construction is successful. We converted the plasmid to ''E. coli'' BL21(DE3) for expression and purification.<br>

Revision as of 12:23, 21 September 2019


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

This part consists of Tac promoter,RBS and protein coding sequence (GST+Linker e+NDM-23),and the biological module can be built into E.coli for protein expression.


Sequence and Features


Assembly Compatibility:
  • 10
    INCOMPATIBLE WITH RFC[10]
    Illegal PstI site found at 874
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal PstI site found at 874
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    INCOMPATIBLE WITH RFC[23]
    Illegal PstI site found at 874
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal PstI site found at 874
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal SapI.rc site found at 181


Usage and Biology

This composite part is made up with three basic parts, the GST tag, the cutting site of Prescission Protease and our target protein Myxo. 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.

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.