Latest revision as of 20:19, 1 November 2017

EF-Tu optimized for expression in E. coli

EF-Tu (elongation factor thermo unstable) is a prokaryotic elongation factor which catalyzes the binding of an aminoacyl-tRNA (aa-tRNA) to the ribosome. It is a G-protein, and is involved in the selection and binding of an aa-tRNA to the A-site of the ribosome. As a result of its crucial role in translation, EF-Tu is one of the most abundant and highly conserved proteins in prokaryotes. Codon optimized for use in Escherichia coli and contains an N-terminal hexahistidine tag. Under the regulation of T7 Promoter, RBS and double terminator.

Improved Part

Original part:BBa_K1906004 submitted by XJTLU-China 2016

Rational behind improvements: BBa_K1906004 encodes exclusively for the coding region of EF-Tu. 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 N-terminal hexahistidine tag with a serine glycine linker for easy purification. We have also optimized EF-Tu 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). The expected size of EF-Tu is 45 kDa, which corresponds to one of the thick bands seen in lane 9 of the overexpression gel below. Overexpression was performed in BL21DE3 gold cells containing K2443027 in pUC57-kan. Overexpressions were performed according to the protocols and notebook on the 2017 iGEM Lethbridge wiki (https://static.igem.org/mediawiki/2017/2/2c/Protein_Overexpression_.pdf and https://static.igem.org/mediawiki/2017/0/0d/October_.pdf)

Sequence and Features