Difference between revisions of "Part:BBa K2933275"

 
 
(4 intermediate revisions by the same user not shown)
Line 3: Line 3:
 
<partinfo>BBa_K2933275 short</partinfo>
 
<partinfo>BBa_K2933275 short</partinfo>
  
−
This part consists of RBS, protein coding sequence(His+Linker h+Sumo+Linker f+JOHN-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.
+
This part consists of RBS, Linker h, protein coding sequence(His+Linker f+JOHN-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.
  
  
Line 18: Line 18:
 
<partinfo>BBa_K2933275 parameters</partinfo>
 
<partinfo>BBa_K2933275 parameters</partinfo>
 
<!-- -->
 
<!-- -->
 +
===Usage and Biology===
 +
This composite part is made up with six basic parts(RBS ,  Linker h, His, Linker f ,  JOHN-1 and T7 terminator ). It encodes a protein which is JOHN-1 fused with His tag. The fusion protein is about 28.1 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.<br>
 +
<p style="text-align: center;">
 +
  [[File:JOHN-1-6p.jpg|600px]]<br>
 +
'''Figure 1.'''  Left: The PCR result of JOHN-1. Right: The verification results by 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>
 +
===References===
 +
[1]Naas Thierry,Bellais Samuel,Nordmann Patrice. Molecular and biochemical characterization of a carbapenem-hydrolysing beta-lactamase from Flavobacterium johnsoniae.[J]. Journal of Antimicrobial Chemotherapy,2003,51(2).<br>

Latest revision as of 14:18, 24 September 2019


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

This part consists of RBS, Linker h, protein coding sequence(His+Linker f+JOHN-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 PstI site found at 204
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal NheI site found at 93
    Illegal NheI site found at 883
    Illegal PstI site found at 204
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    INCOMPATIBLE WITH RFC[23]
    Illegal PstI site found at 204
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal PstI site found at 204
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal SapI.rc site found at 728


Usage and Biology

This composite part is made up with six basic parts(RBS , Linker h, His, Linker f , JOHN-1 and T7 terminator ). It encodes a protein which is JOHN-1 fused with His tag. The fusion protein is about 28.1 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.

JOHN-1-6p.jpg
Figure 1. Left: The PCR result of JOHN-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.

References

[1]Naas Thierry,Bellais Samuel,Nordmann Patrice. Molecular and biochemical characterization of a carbapenem-hydrolysing beta-lactamase from Flavobacterium johnsoniae.[J]. Journal of Antimicrobial Chemotherapy,2003,51(2).