Difference between revisions of "Part:BBa K2933206"
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<partinfo>BBa_K2933206 parameters</partinfo> | <partinfo>BBa_K2933206 parameters</partinfo> | ||
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| − | ==Usage and Biology | + | ==Usage and Biology== |
| − | This composite part is made up with seven basic parts, the His tag, T7 promoter, RBS and our target proteinAFM-1 NDM-23. It encodes a protein which is AFM-1 fused with His tag. The fusion protein is about 28.2 kD. In order to gain the highly purified target protein, we add His tag in N-terminal of AFM-1. It is convenient for us to purify our target protein.<br> | + | This composite part is made up with seven basic parts,the His tag, T7 promoter, RBS ，Linker h ，Linker f，T7 terminator and our target proteinAFM-1 NDM-23. It encodes a protein which is AFM-1 fused with His tag.Linker h is from the vector pET-28a, which connects the RBS to His tag sequence.Linker f from vector pGEX-6p-1, contains the thrombin restriction site and T7 tag. The fusion protein is about 28.2 kD. In order to gain the highly purified target protein, we add His tag in N-terminal of AFM-1. It is convenient for us to purify our target protein.<br> |
===Molecular cloning=== | ===Molecular cloning=== | ||
| − | + | First, we used the vector pGEX-28a 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:AFM-1-PCR1.png|500px]]<br> | [[File:AFM-1-PCR1.png|500px]]<br> | ||
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</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]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 14:01, 23 September 2019
T7 promoter+RBS b+Linker h+His+Linker f+AFM-1+T7 terminator
This part consists of T7 promoter, RBS and protein coding sequence（His+Linker f+AFM-1），and the biological module can be built into E.coli for protein expression.
Sequence and Features
- 10INCOMPATIBLE WITH RFC[10]Illegal XbaI site found at 47
- 12INCOMPATIBLE WITH RFC[12]Illegal NheI site found at 169
- 21COMPATIBLE WITH RFC[21]
- 23INCOMPATIBLE WITH RFC[23]Illegal XbaI site found at 47
- 25INCOMPATIBLE WITH RFC[25]Illegal XbaI site found at 47
- 1000COMPATIBLE WITH RFC[1000]
Usage and Biology
This composite part is made up with seven basic parts,the His tag, T7 promoter, RBS ，Linker h ，Linker f，T7 terminator and our target proteinAFM-1 NDM-23. It encodes a protein which is AFM-1 fused with His tag.Linker h is from the vector pET-28a, which connects the RBS to His tag sequence.Linker f from vector pGEX-6p-1, contains the thrombin restriction site and T7 tag. The fusion protein is about 28.2 kD. In order to gain the highly purified target protein, we add His tag in N-terminal of AFM-1. It is convenient for us to purify our target protein.
Molecular cloning
First, we used the vector pGEX-28a to construct our expression plasmid. And then we converted the plasmid constructed to E. coli DH5α to expand the plasmid largely.
Figure 1. The PCR result of AFM-1.
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.