Difference between revisions of "Part:BBa K2933257"
 
(2 intermediate revisions by the same user not shown)
Line 19: Line 19:
  
 
===Usage and Biology===
 
===Usage and Biology===
−
The Tripoli metallo-beta-lactamase-1 (TMB-1) gene was first discovered in a Achromobacter xylosoxidans strain obtained from an environmental sample in a hospital in Tripoli, Libya, in 2011
+
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 TMB-2 and T7 terminator. It encodes a protein which is TMB-2 fused with His-Sumo tag. The fusion protein is about 39.2 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.
−
After the initial report, TMB-1 has been identified in clinical isolates of Acinetobacter spp. in Japan, and the new TMB-1 variant named TMB-2, with the single mutation S228P, was isolated from a different hospital in Japan also in clinical isolates of Acinetobacter spp.<br>
+
−
 
+
 
===Molecular cloning===
 
===Molecular cloning===
−
First, we used the vector pGEX-6p-1 and pET-28b_SUMO 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 pET-28bs to construct our expression plasmid. And then we converted the plasmid constructed to E. coli DH5α to expand the plasmid largely.
 
<p style="text-align: center;">
 
<p style="text-align: center;">
−
   [[File:TMB-2-PCR.png|400px]]<br>
+
   [[File:TMB-2-PCR--.png|400px]]<br>
 
'''Figure 1.'''  Left: The PCR result of TMB-2. Right: The verification results by enzyme digestion.<br>
 
'''Figure 1.'''  Left: The PCR result of TMB-2. Right: The verification results by enzyme digestion.<br>
 
</p>
 
</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>
 
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]Structural Insights into TMB-1 and the Role of Residues 119 and 228 in Substrate and Inhibitor Binding.Skagseth S, Christopeit T, Akhter S, Bayer A, Samuelsen Ø, Leiros HS.Antimicrob Agents Chemother. 2017 Jul 25;61(8).

Latest revision as of 15:00, 25 September 2019


RBS b+Linker h+His+Linker a+Sumo+Linker b+TMB-2+T7 terminator

This part consists of RBS, protein coding sequence(His+Linker a+Sumo+Linker b+TMB-2) 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
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal EcoRI site found at 298
    Illegal NheI site found at 75
    Illegal NheI site found at 1143
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal EcoRI site found at 298
    Illegal BglII site found at 187
    Illegal BamHI site found at 386
  • 23
    INCOMPATIBLE WITH RFC[23]
    Illegal EcoRI site found at 298
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal EcoRI site found at 298
  • 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 TMB-2 and T7 terminator. It encodes a protein which is TMB-2 fused with His-Sumo tag. The fusion protein is about 39.2 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.

TMB-2-PCR--.png
Figure 1. Left: The PCR result of TMB-2. 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]Structural Insights into TMB-1 and the Role of Residues 119 and 228 in Substrate and Inhibitor Binding.Skagseth S, Christopeit T, Akhter S, Bayer A, Samuelsen Ø, Leiros HS.Antimicrob Agents Chemother. 2017 Jul 25;61(8).