Difference between revisions of "Part:BBa K2933246"

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===Molecular cloning===
 
===Molecular cloning===
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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>
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First, we used the vector pET-28bs 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;">
 
<p style="text-align: center;">
 
   [[File:TJUSLS China--Elbla2-1-PCR.png]]<br>
 
   [[File:TJUSLS China--Elbla2-1-PCR.png]]<br>

Revision as of 11:31, 22 September 2019


RBS b+Linker h+His+Linker a+Sumo+Linker b+ElBlaII+T7 terminator

This part consists of RBS, protein coding sequence(His+Linker a+Sumo+Linker b+ElBla2-1) and T7 terminator,and 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 EcoRI site found at 298
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal EcoRI site found at 298
    Illegal NheI site found at 75
    Illegal NheI site found at 1191
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal EcoRI site found at 298
    Illegal BglII site found at 187
    Illegal BamHI site found at 386
  • 23
    INCOMPATIBLE WITH RFC[23]
    Illegal EcoRI site found at 298
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal EcoRI site found at 298
    Illegal AgeI site found at 881
  • 1000
    COMPATIBLE WITH RFC[1000]


Usage and Biology

This composite part is made up with three basic parts, the His-Sumo tag, the cutting site of Prescission Protease and our target protein ElBla2-1. It encodes a protein which is ElBla2-1 fused with His-Sumo tag. The fusion protein is about 39.5 kD. In order to gain the highly purified target protein, we add GST tag in N-terminal of ElBla2-1 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 pET-28bs to construct our expression plasmid. And then we converted the plasmid constructed to E. coli DH5α to expand the plasmid largely.

TJUSLS China--Elbla2-1-PCR.png
Figure 1. Left: The PCR result of Elbla2-1. 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.

Expression and purification

Pre-expression:
The bacteria were cultured in 5mL LB liquid medium with ampicillin(100 μg/mL final concentration) in 37℃ overnight.