Difference between revisions of "Part:BBa K2933207"
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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 Myxo. It encodes a protein which is Myxo fused with His tag. The fusion protein is about 27.9 kD. In order to gain the highly purified target protein, we add His tag in N-terminal of Myxo . 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 Myxo. It encodes a protein which is Myxo 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 27.9 kD. In order to gain the highly purified target protein, we add His tag in N-terminal of Myxo . 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> | ||
Revision as of 14:01, 23 September 2019
T7 promoter+RBS b+Linker h+His+Linker f+MYX-1+T7 terminator
This part consists of T7 promoter, RBS and protein coding sequence（His+Linker f+Myxo），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
Illegal PstI site found at 284 - 12INCOMPATIBLE WITH RFC[12]Illegal NheI site found at 169
Illegal PstI site found at 284 - 21COMPATIBLE WITH RFC[21]
- 23INCOMPATIBLE WITH RFC[23]Illegal XbaI site found at 47
Illegal PstI site found at 284 - 25INCOMPATIBLE WITH RFC[25]Illegal XbaI site found at 47
Illegal PstI site found at 284 - 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 Myxo. It encodes a protein which is Myxo 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 27.9 kD. In order to gain the highly purified target protein, we add His tag in N-terminal of Myxo . 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 MYX-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]Yabuuchi E, Kaneko T, Yano I, Moss CW, Miyoshi N. Sphingobacterium gen. nov., Sphingobacterium spiritivorum comb. nov., Sphingobacterium multivorum comb. nov., Sphingobacterium mizutae sp. nov., and Flavobacterium indologenes sp. nov.: glucose-nonfermenting gram-negative rods in CDC groups IIK-2 and IIb. Int J Syst Bacteriol. 1983;33:580–98.
[2]Chang Y-C, Lo H-H, Hsieh H-Y, Chang S-M. Identification, epidemiological relatedness, and biofilm formation of clinical Chryseobacterium indologenes isolates from central Taiwan. J Microbiol Immunol Infect. 2015;48:559–64.
[3]Chen F-L, Wang G-C, Teng S-O, Ou T-Y, Yu F-L, Lee W-S. Clinical and epidemiological features of Chryseobacterium indologenes infections: analysis of 215 cases. J Microbiol Immunol Infect. 2013;46:425–32.
[4]Bebrone C. Metallo-beta-lactamases (classification, activity, genetic organization, structure, zinc coordination) and their superfamily. Biochem Pharmacol. 2007;74:1686–701.
[5]Zeba B, De Luca F, Dubus A, Delmarcelle M, Simporé J, Nacoulma OG, et al. IND-6, a highly divergent IND-type metallo-beta-lactamase from Chryseobacterium indologenes strain 597 isolated in Burkina Faso. Antimicrob Agents Chemother. 2009;53:4320–6.
[6]Yamaguchi Y, Takashio N, Wachino J, Yamagata Y, Arakawa Y, Matsuda K, et al. Structure of metallo-beta-lactamase IND-7 from a Chryseobacterium indologenes clinical isolate at 1.65-A resolution. J Biochem. 2010;147:905–15.
[7]Perilli M, Caporale B, Celenza G, Pellegrini C, Docquier JD, Mezzatesta M, et al. Identification and characterization of a new metallo-beta-lactamase, IND-5, from a clinical isolate of Chryseobacterium indologenes. Antimicrob Agents Chemother. 2007;51:2988–90.
[8]Bellais S, Léotard S, Poirel L, Naas T, Nordmann P. Molecular characterization of a carbapenem-hydrolyzing beta-lactamase from Chryseobacterium (Flavobacterium) indologenes. FEMS Microbiol Lett. 1999;171:127–32.