Difference between revisions of "Part:BBa K2443027"
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<partinfo>BBa_K2443027 short</partinfo> | <partinfo>BBa_K2443027 short</partinfo> | ||
| − | 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 <i>Escherichia coli</i> and contains an N-terminal | + | 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 <i>Escherichia coli</i> and contains an N-terminal hexahistidine tag. Under the regulation of T7 Promoter, RBS and double terminator. |
<h1> Improved Part </h1> | <h1> Improved Part </h1> | ||
<p><b>Original part:</b>BBa_K1906004 submitted by XJTLU-China 2016</p> | <p><b>Original part:</b>BBa_K1906004 submitted by XJTLU-China 2016</p> | ||
| − | <p><b> Rational behind improvements:</b> 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). </p> | + | <p><b> Rational behind improvements:</b> 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) </p> |
| + | https://static.igem.org/mediawiki/parts/f/fd/Lethbridge_oct_15_gel_1.png | ||
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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
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]