Difference between revisions of "Part:BBa K2933211"
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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 ，Linker h ，Linker f，T7 terminatorand and our target protein MUS-2. It encodes a protein which is MUS-2 fused with His tag. The fusion protein is about 28.3 kD. In order to gain the highly purified target protein, we add His tag in N-terminal of MUS-2 . 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 terminatorand and our target protein MUS-2. It encodes a protein which is MUS-2 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 28.3 kD. In order to gain the highly purified target protein, we add His tag in N-terminal of MUS-2 . 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> | 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> | ||
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</p> | </p> | ||
After verification, it was determined that the construction is successful. | After verification, it was determined that the construction is successful. | ||
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==References== | ==References== | ||
[1]Al-Bayssari C, Gupta SK, Dabboussi F, Hamze M, Rolain JM. MUS-2, a novel variant of the chromosome-encoded β-lactamase MUS-1, from Myroides odoratimimus. New Microbes New Infect. 2015 Jun 27;7:67-71. | [1]Al-Bayssari C, Gupta SK, Dabboussi F, Hamze M, Rolain JM. MUS-2, a novel variant of the chromosome-encoded β-lactamase MUS-1, from Myroides odoratimimus. New Microbes New Infect. 2015 Jun 27;7:67-71. | ||
[2] Mammeri H, Bellais S, Nordmann P. Chromosome-encoded beta-lactamases TUS-1 and MUS-1 from Myroides odoratus and Myroides odoratimimus (formerly Flavobacterium odoratum), new members of the lineage of molecular subclass B1 metalloenzymes. Antimicrob Agents Chemother. 2002 Nov;46(11):3561-7. | [2] Mammeri H, Bellais S, Nordmann P. Chromosome-encoded beta-lactamases TUS-1 and MUS-1 from Myroides odoratus and Myroides odoratimimus (formerly Flavobacterium odoratum), new members of the lineage of molecular subclass B1 metalloenzymes. Antimicrob Agents Chemother. 2002 Nov;46(11):3561-7. | ||
Latest revision as of 14:02, 23 September 2019
T7 promoter+RBS b+Linker h+His+Linker f+MUS-2+T7 terminator
This part consists of T7 promoter, RBS and protein coding sequence（His+Linker f+MUS-2），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 terminatorand and our target protein MUS-2. It encodes a protein which is MUS-2 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 28.3 kD. In order to gain the highly purified target protein, we add His tag in N-terminal of MUS-2 . 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 MUS-2.
After verification, it was determined that the construction is successful.
References
[1]Al-Bayssari C, Gupta SK, Dabboussi F, Hamze M, Rolain JM. MUS-2, a novel variant of the chromosome-encoded β-lactamase MUS-1, from Myroides odoratimimus. New Microbes New Infect. 2015 Jun 27;7:67-71.
[2] Mammeri H, Bellais S, Nordmann P. Chromosome-encoded beta-lactamases TUS-1 and MUS-1 from Myroides odoratus and Myroides odoratimimus (formerly Flavobacterium odoratum), new members of the lineage of molecular subclass B1 metalloenzymes. Antimicrob Agents Chemother. 2002 Nov;46(11):3561-7.