Difference between revisions of "Part:BBa K2933208"

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T7 promoter+RBS b+Linker h+His+Linker f+IND-10+T7 terminator

This part consists of T7 promoter, RBS and protein coding sequence（His+Linker f+IND-10），and the biological module can be built into E.coli for protein expression.

Sequence and Features

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 IND-10. It encodes a protein which is IND-10 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 26.9 kD. In order to gain the highly purified target protein, we add His tag in N-terminal of IND-10 . 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 IND-10.

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.
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[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.