Difference between revisions of "Part:BBa K2933125"
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<partinfo>BBa_K2933125 parameters</partinfo> | <partinfo>BBa_K2933125 parameters</partinfo> | ||
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| + | ===Usage and Biology=== | ||
| + | This composite part is made up with four 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 40.5kD. 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.<br> | ||
| + | ===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> | ||
| + | <p style="text-align: center;"> | ||
| + | [[File:T--TJUSLS China--MYX-1 PCRmeiqie.png|300px]] [[File:T--TJUSLS China--MYX-1 meiqie.png|300px]]<br> | ||
| + | '''Figure 1.''' Left: The PCR result of MYX-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]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> | ||
Revision as of 13:25, 24 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
- 10INCOMPATIBLE WITH RFC[10]Illegal EcoRI site found at 256
Illegal PstI site found at 435 - 12INCOMPATIBLE WITH RFC[12]Illegal EcoRI site found at 256
Illegal NheI site found at 33
Illegal PstI site found at 435 - 21INCOMPATIBLE WITH RFC[21]Illegal EcoRI site found at 256
Illegal BglII site found at 145
Illegal BamHI site found at 344 - 23INCOMPATIBLE WITH RFC[23]Illegal EcoRI site found at 256
Illegal PstI site found at 435 - 25INCOMPATIBLE WITH RFC[25]Illegal EcoRI site found at 256
Illegal PstI site found at 435 - 1000COMPATIBLE WITH RFC[1000]
Usage and Biology
This composite part is made up with four 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 40.5kD. 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 pGEX-6p-1 to construct our expression plasmid. And then we converted the plasmid constructed to E. coli DH5α to expand the plasmid largely.
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.