Difference between revisions of "Part:BBa K4361313"

 
 
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A mutant of the BlcR protein, created through site-directed mutagenesis with primers R3 ([[Part:BBa_K4361218]]) and F3.4 A67Q ([[Part:BBa_K4361222]]). For this mutant, the alanine in position 67 has been changed to glutamine by mutating the GCG codon to CAG.
 
A mutant of the BlcR protein, created through site-directed mutagenesis with primers R3 ([[Part:BBa_K4361218]]) and F3.4 A67Q ([[Part:BBa_K4361222]]). For this mutant, the alanine in position 67 has been changed to glutamine by mutating the GCG codon to CAG.
  
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This mutant also contains the following nucleotide mutations outside of the targeted site:
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This mutant contains no nucleotide substitutions or indels outside of the targeted site.
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<span class='h3bb'>Sequence and Features</span>
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<span class='h3bb'><h3>Sequence and Features</h3></span>
 
<partinfo>BBa_K4361313 SequenceAndFeatures</partinfo>
 
<partinfo>BBa_K4361313 SequenceAndFeatures</partinfo>
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<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 14</u>), a protein band corresponding to the BlcR monomer is visible. Concluding that the production in an E. coli cell-free system was successful for this mutant.
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<a href="https://static.igem.wiki/teams/4361/wiki/results/figure-7-pure/figure-7-pure-14.png"><img src="https://static.igem.wiki/teams/4361/wiki/results/figure-7-pure/figure-7-pure-14.png" style="width:600px;margin-left:125px"></a>
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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. 14: A67Q mutant.</figcaption>
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Latest revision as of 15:56, 12 October 2022


BlcR A67Q

A mutant of the BlcR protein, created through site-directed mutagenesis with primers R3 (Part:BBa_K4361218) and F3.4 A67Q (Part:BBa_K4361222). For this mutant, the alanine in position 67 has been changed to glutamine by mutating the GCG codon to CAG.

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 14), a protein band corresponding to the BlcR monomer is visible. Concluding that the production in an E. coli cell-free system was successful for this mutant.
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. 14: A67Q mutant.