Difference between revisions of "Part:BBa K2933126"

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(→‎Usage and Biology)
 
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===Usage and Biology===
 
===Usage and Biology===
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This composite part is made up with four basic parts, the His tag, the Sumo tag, three cutting sites(NdeI, NheI and BamHI) and our target protein IND-10. It encodes a protein which is IND-10 fused with His-Sumo tag. The fusion protein is about 38.9kD. In order to gain the highly purified target protein, we add His-Sumo tag in N-terminal of IND-10 and combine the three parts with these three cutting sites. The fusion protein can be cut off at the cutting site BamHI. It is convenient for us to purify our target protein.<br>
+
This composite part is made up with five basic parts, the His tag, the Sumo tag, three cutting sites(NdeI, NheI and BamHI) and our target protein IND-10. It encodes a protein which is IND-10 fused with His-Sumo tag. The fusion protein is about 38.9kD. In order to gain the highly purified target protein, we add His-Sumo tag in N-terminal of IND-10 and combine the three parts with these three cutting sites. The fusion protein can be cut off at the cutting site BamHI. It is convenient for us to purify our target protein.<br>
  
 
===References===
 
===References===

Latest revision as of 11:29, 25 September 2019


His+Linker a+Sumo+Linker b+IND-10

This part encodes the fusion protein of His tag, sumo tag and IND-10 to promote the expression and purification of target protein(IND-10).


Sequence and Features


Assembly Compatibility:
  • 10
    INCOMPATIBLE WITH RFC[10]
    Illegal EcoRI site found at 256
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal EcoRI site found at 256
    Illegal NheI site found at 33
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal EcoRI site found at 256
    Illegal BglII site found at 145
    Illegal BamHI site found at 344
  • 23
    INCOMPATIBLE WITH RFC[23]
    Illegal EcoRI site found at 256
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal EcoRI site found at 256
  • 1000
    COMPATIBLE WITH RFC[1000]


Usage and Biology

This composite part is made up with five basic parts, the His tag, the Sumo tag, three cutting sites(NdeI, NheI and BamHI) and our target protein IND-10. It encodes a protein which is IND-10 fused with His-Sumo tag. The fusion protein is about 38.9kD. In order to gain the highly purified target protein, we add His-Sumo tag in N-terminal of IND-10 and combine the three parts with these three cutting sites. The fusion protein can be cut off at the cutting site BamHI. 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 pET28b-sumo to construct our expression plasmid. And then we converted the plasmid constructed to E. coli DH5α to expand the plasmid largely.

T--TJUSLS China--IND-10 PCR.png T--TJUSLS China--IND-10 PCRmeiqie.png
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.