Part:BBa_K2443028

EF-Ts optimized for expression in E. coli

EF-Ts is a prokaryotic elongation factors. EF-Ts acts at the guanine nucleotide exchange factor for EF-Tu, catalyzing the release of guanosine diphosphate from EF-Tu. This enables EF-Tu to bind to a new guanosine triphosphate molecule, release EF-Ts, and go on to catalyze another aminoacyl tRNA addition. EF-Ts has been Codon optimized for use in Escherichia coli and contains a C-terminal hexahistidine tag. Under the regulation of T7 Promoter, RBS and double terminator. The expected size of EF-Ts is 32 kDa, corresponding to the band observed lane 9. Protein overexpression and purification was performed using BL21DE3 gold cells containing pJET with K2443028 inserted. Experiments were performed according to the Lethbridge 2017 protocols and notebook found on the wiki (https://static.igem.org/mediawiki/2017/2/2c/Protein_Overexpression_.pdf and https://static.igem.org/mediawiki/2017/0/0d/October_.pdf).

Improved Part

Original part:BBa_K1906000 submitted by XJTLU-China 2016

Rational behind improvements: BBa_K1906000 encodes exclusively for the coding region of EF-Ts. In order to improve it and incorporate it into our Next vivo system we have codon optimized for use in E. coli and attached a C-terminal hexahistidine tag with a serine glycine linker for easy purification. We have also optimized EF-Ts to be overexpressed in BL21-Gold (DE3) cells by putting it under the control of a T7 promoter (BBa_I719005), RBS (BBa_B0034) and double terminator (BBa_B0015).

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal NheI site found at 65
  
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  COMPATIBLE WITH RFC[1000]