Difference between revisions of "Part:BBa K2933242"

 
 
Line 17: Line 17:
 
<partinfo>BBa_K2933242 parameters</partinfo>
 
<partinfo>BBa_K2933242 parameters</partinfo>
 
<!-- -->
 
<!-- -->
 +
===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 Fla.103. It encodes a protein which is Fla.103 fused with GST tag. The fusion protein is about 54.7 kD. In order to gain the highly purified target protein, we add GST tag in N-terminal of Fla.103 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.We then extracted plasmids and performed double enzyme digestion verification.<br>
 +
<p style="text-align: center;">
 +
  [[File:Fla.103-PCR.png]]<br>
 +
'''Figure 1.'''  (a) The PCR result of Fla.103. (b) The verification results by enzyme digestion.The 1 is the original plasmid.The 2 is the
 +
results verified by double 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>

Latest revision as of 14:02, 23 September 2019


RBS a+Linker g+GST+Linker e+Fla.103

This part consists of RBS a, protein coding sequence(GST+Linker e+Fla.103). The blaFla.103 is separated from Flavobacterium sp. 103. 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 802
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal PstI site found at 802
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BglII site found at 1284
  • 23
    INCOMPATIBLE WITH RFC[23]
    Illegal PstI site found at 802
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal PstI site found at 802
  • 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 Fla.103. It encodes a protein which is Fla.103 fused with GST tag. The fusion protein is about 54.7 kD. In order to gain the highly purified target protein, we add GST tag in N-terminal of Fla.103 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.We then extracted plasmids and performed double enzyme digestion verification.

Fla.103-PCR.png
Figure 1. (a) The PCR result of Fla.103. (b) The verification results by enzyme digestion.The 1 is the original plasmid.The 2 is the results verified by double 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.