Difference between revisions of "Part:BBa K2933213"
(→‎Usage and Biology=)
Line 19: Line 19:
 
<!-- -->
 
<!-- -->
 
==Usage and Biology===
 
==Usage and Biology===
−
This composite part is made up with three basic parts, the His tag, T7 promoter, RBS and our target protein JOHN-1. It encodes a protein which is JOHN-1 fused with His tag. The fusion protein is about 28.1 kD. In order to gain the highly purified target protein, we add His tag in N-terminal of JOHN-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 JOHN-1. It encodes a protein which is JOHN-1 fused with His tag. The fusion protein is about 28.1 kD. In order to gain the highly purified target protein, we add His tag in N-terminal of JOHN-1 .  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 13:00, 23 September 2019


T7 promoter+RBS b+Linker h+His+Linker f+JOHN-1+T7 terminator

This part consists of T7 promoter, RBS and protein coding sequence（His+Linker f+JOHN-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
    Illegal PstI site found at 280
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal NheI site found at 169
    Illegal PstI site found at 280
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    INCOMPATIBLE WITH RFC[23]
    Illegal XbaI site found at 47
    Illegal PstI site found at 280
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal XbaI site found at 47
    Illegal PstI site found at 280
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal SapI.rc site found at 804


Usage and Biology=

This composite part is made up with seven basic parts, the His tag, T7 promoter, RBS and our target protein JOHN-1. It encodes a protein which is JOHN-1 fused with His tag. The fusion protein is about 28.1 kD. In order to gain the highly purified target protein, we add His tag in N-terminal of JOHN-1 . 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.

JOHN-1-6p1.jpg
Figure 1. The PCR result of JOHN-1.

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