Part:BBa_K2933228

RBS a+Linker g+GST+Linker e+IND-10

This part consists of RBS a, protein coding sequence(GST+Linker e+IND-10), the RBS and the protein coding sequence can be connected by linker g. The biological module can be build into E.coli for protein expression. This part can be prefaced with promoters of different strengths and types to regulate expression function.

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal SapI.rc site found at 113
  

Usage and Biology

This composite part is made up with five basic parts, the RBSa, the linker g and the GST tag, the cutting site of Prescission Protease and our target protein IND-10. It encodes a protein which is IND-10 fused with GST tag. The fusion protein is about 52.9 kD. In order to gain the highly purified target protein, we add GST tag in N-terminal of IND-10 and combine the two parts with the cutting site of Prescission Protease. The fusion protein can be cut off at the cutting site by Prescission Protease. It is convenient for us to purify our target protein.

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.

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 IND-10. 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.