Difference between revisions of "Part:BBa K2933263"
Line 18: Line 18:
 
<!-- -->
 
<!-- -->
 
===Usage and Biology===
 
===Usage and Biology===
−
Fla.103 is a type of subclass B metal beta-lactamases. The beta lactamases can hydrolyze almost all available beta lactam antibiotics (except aztreonam) clinically, including the broad-spectrum antibiotic carbapenems. Because of the extensive substrate profile of this enzyme, the clinical strains carrying it become a great threat to human life and health.<br>
+
This composite part is made up with eight basic parts, T7 Ribosome binding sites,the His-Sumo tag, three cutting sites of Prescission Protease, our target protein Fla.103 and T7 terminator. It encodes a protein which is Fla.103 fused with His-Sumo tag. The fusion protein is about 40.7 kD. The fusion protein can be cut off at the cutting sites by Prescission Protease. It is convenient for us to purify our target protein.
−
 
+
Molecular cloning
−
===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.
−
 
+
−
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.We then extracted plasmids and performed double enzyme digestion verification.<br>
+
 
<p style="text-align: center;">
 
<p style="text-align: center;">
 
   [[File:Fla.103-PCR.png]]<br>
 
   [[File:Fla.103-PCR.png]]<br>

Revision as of 14:05, 22 September 2019


RBS b+Linker h+His+Linker a+Sumo+Linker b+Fla.103+T7 terminator

This part consists of RBS, protein coding sequence(His+Linker a+Sumo+Linker b+Fla.103) 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
    Illegal PstI site found at 473
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal EcoRI site found at 298
    Illegal NheI site found at 75
    Illegal NheI site found at 1158
    Illegal PstI site found at 473
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal EcoRI site found at 298
    Illegal BglII site found at 187
    Illegal BglII site found at 955
    Illegal BamHI site found at 386
  • 23
    INCOMPATIBLE WITH RFC[23]
    Illegal EcoRI site found at 298
    Illegal PstI site found at 473
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal EcoRI site found at 298
    Illegal PstI site found at 473
  • 1000
    COMPATIBLE WITH RFC[1000]


Usage and Biology

This composite part is made up with eight basic parts, T7 Ribosome binding sites,the His-Sumo tag, three cutting sites of Prescission Protease, our target protein Fla.103 and T7 terminator. It encodes a protein which is Fla.103 fused with His-Sumo tag. The fusion protein is about 40.7 kD. The fusion protein can be cut off at the cutting sites 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.

Fla.103-PCR.png
Figure 1. (a) The PCR result of Fla.103. (b) The verification results by enzyme digestion.The 1 is the original plasmid.The 2 is the results verified by double 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.