Difference between revisions of "Part:BBa K4361306"
 
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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 7</u>), a band was visible on the gel at the expected position, meaning that the production was successful for this mutant.
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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 7</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-7.png"><img src="https://static.igem.wiki/teams/4361/wiki/results/figure-7-pure/figure-7-pure-7.png" style="width:600px;margin-left:125px"></a>
 
<a href="https://static.igem.wiki/teams/4361/wiki/results/figure-7-pure/figure-7-pure-7.png"><img src="https://static.igem.wiki/teams/4361/wiki/results/figure-7-pure/figure-7-pure-7.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. 7: A62K mutant.</figcaption>
 
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Latest revision as of 18:18, 13 October 2022


BlcR A62K

A mutant of the BlcR protein, created through site-directed mutagenesis with primers R7 (Part:BBa_K4361210) and F7.4 A62K (Part:BBa_K4361214). For this mutant, the alanine in position 62 has been changed to lysine by mutating the GCG codon to AAA.

This mutant also contains the following nucleotide mutations outside of the targeted site:

  • A 800 deletion, resulting in a possible frameshift and extension of the protein

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 7), 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. 7: A62K mutant.