Difference between revisions of "Part:BBa K1906004"
 
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<P>It was quite surprising that plasmids that carry beta subunit genes don't lead to a heavy production of the protein upon the induction. However, this situation can be improved when EF-Ts and EF-Tu are co-expressed with beta subunit.</P>
 
<P>It was quite surprising that plasmids that carry beta subunit genes don't lead to a heavy production of the protein upon the induction. However, this situation can be improved when EF-Ts and EF-Tu are co-expressed with beta subunit.</P>
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<p>Figure 1 is the SDS-PAGE result comparison between cells that transformed with these three proteins and beta subunit alone.</P>
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<p>Figure 1 is the SDS-PAGE result comparison between cells that were transformed with these three proteins and beta subunit alone.</P>
  
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[[File:20161019150339!SDS-TSTU-REP.jpg|500px|thumb|left|Figure 1.SDS-PAGE result comparison between three-protein-expressing cells and beta subunit-expressing cells, wild-type cells were included as a control group. The gel on the left showing the results of cells that been induced with 2mM IPTG and the gel on the right shows the results of same groups of cells that not been induced. The blue arrow indicates the band position of beta subunit.]]
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[[File:20161019150339!SDS-TSTU-REP.jpg|500px|thumb|left|Figure 1. SDS-PAGE result comparison between three-protein-expressing cells and beta subunit-expressing cells. Wild-type cells were included as a control group. The gel on the left shows the results of cells that had been induced with 2mM IPTG and the gel on the right shows the results of same groups of cells that had not been induced. The blue arrow indicates the band position of beta subunit.]]
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<br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br>
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It can be clearly seen that cells transformed with beta subunit alone had no detectable level of protein expression. However, the expression of beta subunit can be greatly restored when plasmids that carry EF-Tu and EF-Ts are transformed into the cells.
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<h1> Improved Part </h1>
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<p><b>Improved part:</b> BBa_K2443027 submitted by Lethbridge iGEM 2017</p>
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<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) gold cells by putting it under the control of a T7 promoter (BBa_I719005), RBS (BBa_B0034) and double terminator (BBa_B0015).  </p>
  
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It can be clearly seen that cells transformed with beta subunit alone had no detectable level of protein expression. However, the expression of beta subunit can be greatly restored when plasmids carry EF-Tu and EF-Ts are transformed into the cells.
 
  
 
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Latest revision as of 02:06, 31 October 2017


Elongation Factor Tu

This protein is well known as a translation elongation factor in bacterium Escherichia coli. It can also assemble with phage polymerase domain to form an RNA-dependent-RNA polymerase. It was suggested by XJTLU-CHINA 2016 iGEM team that over-expression of this protein together with EF-TS are essential for the production of beta subunit of Qbeta replicase holoenzyme.

Characterlization

It was quite surprising that plasmids that carry beta subunit genes don't lead to a heavy production of the protein upon the induction. However, this situation can be improved when EF-Ts and EF-Tu are co-expressed with beta subunit.

Figure 1 is the SDS-PAGE result comparison between cells that were transformed with these three proteins and beta subunit alone.

Figure 1. SDS-PAGE result comparison between three-protein-expressing cells and beta subunit-expressing cells. Wild-type cells were included as a control group. The gel on the left shows the results of cells that had been induced with 2mM IPTG and the gel on the right shows the results of same groups of cells that had not been induced. The blue arrow indicates the band position of beta subunit.




















It can be clearly seen that cells transformed with beta subunit alone had no detectable level of protein expression. However, the expression of beta subunit can be greatly restored when plasmids that carry EF-Tu and EF-Ts are transformed into the cells.

Improved Part

Improved part: BBa_K2443027 submitted by Lethbridge iGEM 2017

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) gold 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
    COMPATIBLE WITH RFC[12]
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal AgeI site found at 280
    Illegal AgeI site found at 685
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal SapI.rc site found at 430