Difference between revisions of "Part:BBa K4361310"
 
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<h3>Usage and Biology</h3>
 
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The set of BlcR mutants (this part through </html>[[Part:BBa_K4361319]]<html>) were expressed in a PURE system in the presence of GreenLys (fluorescent Cy2-labeled lysine). Samples were then run on an SDS PAGE gel (<b>Figure 1.</b>) to determine whether or not the protein was produced. For this mutant (<u>lane 11</u>), it was concluded that the production was not successful due to the bands corresponding to BlcR being too poorly distinguishable.
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The set of BlcR mutants (</html>[[Part:BBa_K4361300]] through [[Part:BBa_K4361319]]<html>) were expressed in a PURE system in the presence of GreenLys (fluorescent Cy2-labeled lysine). Samples were then run on an SDS PAGE gel (<b>Figure 1.</b>) to determine whether or not the protein was produced. For this mutant (<u>lane 11</u>), production in an <i> E. coli </i> based cell free system was not successful, no bands corresponding to BlcR were visible.
  
 
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<a href="https://static.igem.wiki/teams/4361/wiki/results/figure-7-pure/figure-7-pure-11.png"><img src="https://static.igem.wiki/teams/4361/wiki/results/figure-7-pure/figure-7-pure-11.png" style="width:600px;margin-left:125px"></a>
 
<a href="https://static.igem.wiki/teams/4361/wiki/results/figure-7-pure/figure-7-pure-11.png"><img src="https://static.igem.wiki/teams/4361/wiki/results/figure-7-pure/figure-7-pure-11.png" style="width:600px;margin-left:125px"></a>
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<figcaption> <b>Figure 1.</b> SDS PAGE gel of PURE reactions with GreenLys and a variant of </html>[[Part:BBa_K4361106]]<html> containing a BlcR mutant. Arrows indicate bands corresponding to BlcR dimers and monomers, respectively. C = negative control, WT = wildtype BlcR.</figcaption>
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<figcaption> <b>Figure 1.</b> SDS PAGE showing the translation products of 20 different plasmids coding for BlcR mutants. PURE reaction solution was supplemented with GreenLys and the synthesized proteins were imaged with Typhoon using a 488-nm laser. C: negative control with no plasmid. WT: positive control with expressed wildtype BlcR. 11: L66V mutant.</figcaption>
 
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Latest revision as of 18:21, 13 October 2022


BlcR L66V

A mutant of the BlcR protein, created through site-directed mutagenesis with primers R3 (Part:BBa_K4361218) and F3.1 L66V (Part:BBa_K4361219). For this mutant, the leucine in position 66 has been changed to valine by mutating the CTC codon to GTG.

This mutant contains no nucleotide substitutions or indels outside of the targeted site.

Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal NheI site found at 694
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal NgoMIV site found at 78
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal SapI.rc site found at 589

Usage and Biology

The set of BlcR mutants (Part:BBa_K4361300 through Part:BBa_K4361319) were expressed in a PURE system in the presence of GreenLys (fluorescent Cy2-labeled lysine). Samples were then run on an SDS PAGE gel (Figure 1.) to determine whether or not the protein was produced. For this mutant (lane 11), production in an E. coli based cell free system was not successful, no bands corresponding to BlcR were visible.
Figure 1. SDS PAGE showing the translation products of 20 different plasmids coding for BlcR mutants. PURE reaction solution was supplemented with GreenLys and the synthesized proteins were imaged with Typhoon using a 488-nm laser. C: negative control with no plasmid. WT: positive control with expressed wildtype BlcR. 11: L66V mutant.