Difference between revisions of "Part:BBa K2933125"

(→‎Usage and Biology)
(→‎Molecular cloning)
 
Line 22: Line 22:
  
 
===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 pET28b-sumo 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;">
 
<p style="text-align: center;">
 
   [[File:T--TJUSLS China--MYX-1 PCRmeiqie.png|300px]]  [[File:T--TJUSLS China--MYX-1 meiqie.png|300px]]<br>
 
   [[File:T--TJUSLS China--MYX-1 PCRmeiqie.png|300px]]  [[File:T--TJUSLS China--MYX-1 meiqie.png|300px]]<br>
Line 28: Line 28:
 
</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===
 
===References===
 
[1]Correction for Goldman et al., Evolution of sensory complexity recorded in a myxobacterial genome. Proc Natl Acad Sci U S A. 2006;103(51):19605. doi:10.1073/pnas.0609567103.<br>
 
[1]Correction for Goldman et al., Evolution of sensory complexity recorded in a myxobacterial genome. Proc Natl Acad Sci U S A. 2006;103(51):19605. doi:10.1073/pnas.0609567103.<br>

Latest revision as of 11:28, 25 September 2019


His+Linker a+Sumo+Linker b+MYX-1

This part encodes the fusion protein of His tag, sumo tag and MYX-1 to promote the expression and purification of target protein(MYX-1).


Sequence and Features


Assembly Compatibility:
  • 10
    INCOMPATIBLE WITH RFC[10]
    Illegal EcoRI site found at 256
    Illegal PstI site found at 435
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal EcoRI site found at 256
    Illegal NheI site found at 33
    Illegal PstI site found at 435
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal EcoRI site found at 256
    Illegal BglII site found at 145
    Illegal BamHI site found at 344
  • 23
    INCOMPATIBLE WITH RFC[23]
    Illegal EcoRI site found at 256
    Illegal PstI site found at 435
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal EcoRI site found at 256
    Illegal PstI site found at 435
  • 1000
    COMPATIBLE WITH RFC[1000]


Usage and Biology

This composite part is made up with five basic parts, the His tag, the Sumo tag, three cutting sites(NdeI, NheI and BamHI) and our target protein MYX-1. It encodes a protein which is MYX-1 fused with His-Sumo tag. The fusion protein is about 39.9kD. In order to gain the highly purified target protein, we add His-Sumo tag in N-terminal of MYX-1 and combine the three parts with these three cutting sites. The fusion protein can be cut off at the cutting site BamHI. It is convenient for us to purify our target protein.

Molecular cloning

First, we used the vector pET28b-sumo to construct our expression plasmid. And then we converted the plasmid constructed to E. coli DH5α to expand the plasmid largely.

T--TJUSLS China--MYX-1 PCRmeiqie.png T--TJUSLS China--MYX-1 meiqie.png
Figure 1. Left: The PCR result of MYX-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]Correction for Goldman et al., Evolution of sensory complexity recorded in a myxobacterial genome. Proc Natl Acad Sci U S A. 2006;103(51):19605. doi:10.1073/pnas.0609567103.