Difference between revisions of "Part:BBa K2933267"
(→‎Usage and Biology)
Line 19: Line 19:
 
<!-- -->
 
<!-- -->
 
===Usage and Biology===
 
===Usage and Biology===
−
This composite part is made up with three basic parts(RBS，Linker h and T7 terminator)and a composite part(His+Linker f+ElBla2-1). It encodes a protein which is Elbla2-1 fused with His tag. The fusion protein is about 27.5 kD. It is convenient for us to purify our target protein.
+
This composite part is made up with four basic parts(RBS，Linker h , T7 terminator and His+Linker f+ElBla2-1). It encodes a protein which is Elbla2-1 fused with His tag. The fusion protein is about 27.5 kD. It is convenient for us to purify our target protein.
 +
 
 
===Molecular cloning===
 
===Molecular cloning===
 
First, we used the vector pET28a 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 pET28a 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 16:21, 23 September 2019


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

This part consists of RBS, protein coding sequence(His+Linker h+Sumo+Linker f+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
    COMPATIBLE WITH RFC[10]
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal NheI site found at 93
    Illegal NheI site found at 922
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal AgeI site found at 612
  • 1000
    COMPATIBLE WITH RFC[1000]


Usage and Biology

This composite part is made up with four basic parts(RBS，Linker h , T7 terminator and His+Linker f+ElBla2-1). It encodes a protein which is Elbla2-1 fused with His tag. The fusion protein is about 27.5 kD. It is convenient for us to purify our target protein.

Molecular cloning

First, we used the vector pET28a to construct our expression plasmid. And then we converted the plasmid constructed to E. coli DH5α to expand the plasmid largely.

Elbla pcr.png
Figure 1. The PCR result of Elbla2-1.

References

[1] Girlich D, Poirel L, Nordmann P, Diversity of naturally occurring Ambler class B metallo-β-lactamases in Erythrobacter spp. The Journal of Antimicrobial Chemotherapy [31 Jul 2012, 67(11):2661-2664]