Difference between revisions of "Part:BBa K4361311"
 
(One intermediate revision by one other user not shown)
Line 3: Line 3:
 
<partinfo>BBa_K4361311 short</partinfo>
 
<partinfo>BBa_K4361311 short</partinfo>
  
−
A mutant of the BlcR protein, created through site-directed mutagenesis with primers R3 ([[Part:BBa_K4361218]]) and F3.2 L66V ([[Part:BBa_K4361220]]). For this mutant, the leucine in position 66 has been changed to alanine by mutating the CTC codon to GCG.
+
A mutant of the BlcR protein, created through site-directed mutagenesis with primers R3 ([[Part:BBa_K4361218]]) and F3.2 L66A ([[Part:BBa_K4361220]]). For this mutant, the leucine in position 66 has been changed to alanine by mutating the CTC codon to GCG.
  
 
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:
Line 20: Line 20:
 
<html>
 
<html>
 
<h3>Usage and Biology</h3>
 
<h3>Usage and Biology</h3>
−
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 12</u>), a band was visible on the gel at the expected position, meaning that the production was successful for this mutant.
+
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 12</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.
−
 
+
 
<figure>
 
<figure>
 
<a href="https://static.igem.wiki/teams/4361/wiki/results/figure-7-pure/figure-7-pure-12.png"><img src="https://static.igem.wiki/teams/4361/wiki/results/figure-7-pure/figure-7-pure-12.png" style="width:600px;margin-left:125px"></a>
 
<a href="https://static.igem.wiki/teams/4361/wiki/results/figure-7-pure/figure-7-pure-12.png"><img src="https://static.igem.wiki/teams/4361/wiki/results/figure-7-pure/figure-7-pure-12.png" style="width:600px;margin-left:125px"></a>
−
<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>
+
<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. 12: L66A mutant</figcaption>
 
</figure>
 
</figure>
  

Latest revision as of 15:54, 12 October 2022


BlcR L66A

A mutant of the BlcR protein, created through site-directed mutagenesis with primers R3 (Part:BBa_K4361218) and F3.2 L66A (Part:BBa_K4361220). For this mutant, the leucine in position 66 has been changed to alanine by mutating the CTC codon to GCG.

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

  • T 826 > A, resulting in possible loss of the stop codon

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 12), 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. 12: L66A mutant