Difference between revisions of "Part:BBa K4361300"
 
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<partinfo>BBa_K4361300 short</partinfo>
 
<partinfo>BBa_K4361300 short</partinfo>
  
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A mutant of the BlcR protein, created through site-directed mutagenesis with primers R1 ([[Part:BBa_K4361200]]) and F1.1 D37R ([[Part:BBa_K4361201]]). For this mutant, the aspartic acid in position 37 has been changed to arginine by mutating the GAC codon to CGC.
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A mutant of the BlcR protein ([[Part:BBa_K4361100]]), created through site-directed mutagenesis with primers R1 ([[Part:BBa_K4361200]]) and F1.1 D37R ([[Part:BBa_K4361201]]). For this mutant, the aspartic acid in position 37 has been changed to arginine by mutating the GAC codon to CGC.
  
 
This mutant also contains the following nucleotide mutations outside of the targeted site:
 
This mutant also contains the following nucleotide mutations outside of the targeted site:
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<h3>Usage and Biology</h3>
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<h3>Experimental results</h3>
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The set of BlcR mutants (this part through </html>[[Part:BBa_K4361319]]<html>) were designed to be used in a PURE reaction together with </html>[[Part:BBa_K4361115]] and [[Part:BBa_K4361117]]<html> to translate the mutants and subsequently measure their binding strength to the wildtype Blc operator and a mutant, respectively. As the results of these experiments were inconsistent, it was concluded that PURE was not the correct system to screen BlcR activity. However, through the addition of GreenLys (fluorescent Cy2-labeled lysine), the results from PURE could still be used to show whether or not the mutants were produced in solution (see <b>Figure 1.</b>). For this mutant (<u>lane 1</u>), it was concluded that the production was not succesful due to the bands corresponding to BlcR being too poorly distinguishable.
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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 1</u>), BlcR mutant D37R could not be succesfully produced in the PURE system.
  
 
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<a href="https://static.igem.wiki/teams/4361/wiki/results/greenlys.png"><img src="https://static.igem.wiki/teams/4361/wiki/results/greenlys.png" style="width:600px;margin-left:125px"></a>
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<a href="https://static.igem.wiki/teams/4361/wiki/results/figure-7-pure/figure-7-pure-1.png"><img src="https://static.igem.wiki/teams/4361/wiki/results/figure-7-pure/figure-7-pure-1.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. 1: D37R mutant. </figcaption>
 
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</figure>
  

Latest revision as of 18:14, 13 October 2022


BlcR D37R

A mutant of the BlcR protein (Part:BBa_K4361100), created through site-directed mutagenesis with primers R1 (Part:BBa_K4361200) and F1.1 D37R (Part:BBa_K4361201). For this mutant, the aspartic acid in position 37 has been changed to arginine by mutating the GAC codon to CGC.

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

  • G 763 > A, resulting in substitution E255K

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

Experimental results

The set of BlcR mutants (this part 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 1), BlcR mutant D37R could not be succesfully produced in the PURE 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. 1: D37R mutant.