Difference between revisions of "Part:BBa K4361304"
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<h3>Usage and Biology</h3>
 
<h3>Usage and Biology</h3>
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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 5</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 5</u>), no protein bands corresponding to the BlcR monomer are visible, concluding that the mutant D37V was not successfully produced in the <i> E. coli </i> cell-free system.
  
 
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<a href="https://static.igem.wiki/teams/4361/wiki/results/figure-7-pure/figure-7-pure-5.png"><img src="https://static.igem.wiki/teams/4361/wiki/results/figure-7-pure/figure-7-pure-5.png" style="width:600px;margin-left:125px"></a>
 
<a href="https://static.igem.wiki/teams/4361/wiki/results/figure-7-pure/figure-7-pure-5.png"><img src="https://static.igem.wiki/teams/4361/wiki/results/figure-7-pure/figure-7-pure-5.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. 2: D37V mutant</figcaption>
 
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Revision as of 15:46, 12 October 2022


BlcR A62V

A mutant of the BlcR protein, created through site-directed mutagenesis with primers R7 (Part:BBa_K4361210) and F7.2 A62V (Part:BBa_K4361212). For this mutant, the alanine in position 62 has been changed to valine by mutating the GCG 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 5), no protein bands corresponding to the BlcR monomer are visible, concluding that the mutant D37V was not successfully produced in the E. coli cell-free system.
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. 2: D37V mutant