Difference between revisions of "Part:BBa K2933205"

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(→‎Molecular cloning)
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This composite part is made up with seven basic parts, the His tag, T7 promoter, RBS and our target protein ElBla2-1. It encodes a protein which is ElBla2-1NDM-23 fused with His tag. The fusion protein is about 27.5 kD. In order to gain the highly purified target protein, we add His tag in N-terminal of ElBla2-1 .  It is convenient for us to purify our target protein.<br>
 
This composite part is made up with seven basic parts, the His tag, T7 promoter, RBS and our target protein ElBla2-1. It encodes a protein which is ElBla2-1NDM-23 fused with His tag. The fusion protein is about 27.5 kD. In order to gain the highly purified target protein, we add His tag in N-terminal of ElBla2-1 .  It is convenient for us to purify our target protein.<br>
 
===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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<p style="text-align: center;">
 
<p style="text-align: center;">
 
   [[File:TJUSLS China--Elbla2-1-PCR1.png]]<br>
 
   [[File:TJUSLS China--Elbla2-1-PCR1.png]]<br>

Revision as of 13:05, 23 September 2019


T7 promoter+RBS b+Linker h+His+Linker f+ElBlaII+T7 terminator

This part consists of T7 promoter, RBS and protein coding sequence（His+Linker f+ElBlA2-1），and the biological module can be built into E.coli for protein expression.


Sequence and Features


Assembly Compatibility:
  • 10
    INCOMPATIBLE WITH RFC[10]
    Illegal XbaI site found at 47
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal NheI site found at 169
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    INCOMPATIBLE WITH RFC[23]
    Illegal XbaI site found at 47
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal XbaI site found at 47
    Illegal AgeI site found at 688
  • 1000
    COMPATIBLE WITH RFC[1000]


Usage and Biology=

This composite part is made up with seven basic parts, the His tag, T7 promoter, RBS and our target protein ElBla2-1. It encodes a protein which is ElBla2-1NDM-23 fused with His tag. The fusion protein is about 27.5 kD. In order to gain the highly purified target protein, we add His tag in N-terminal of ElBla2-1 . It is convenient for us to purify our target protein.

Molecular cloning

TJUSLS China--Elbla2-1-PCR1.png
Figure 1. The PCR result of Elbla2-1.

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.