Difference between revisions of "Part:BBa K4361315"
 
(One intermediate revision by the same user not shown)
Line 22: Line 22:
 
<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 16</u>), it was concluded that the production was not successful due to the bands corresponding to BlcR being too poorly distinguishable.
+
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 16</u>), production in an E. coli based cell free system was not successful, no bands corresponding to BlcR were visible.
  
 
<figure>
 
<figure>
 
<a href="https://static.igem.wiki/teams/4361/wiki/results/figure-7-pure/figure-7-pure-16.png"><img src="https://static.igem.wiki/teams/4361/wiki/results/figure-7-pure/figure-7-pure-16.png" style="width:600px;margin-left:125px"></a>
 
<a href="https://static.igem.wiki/teams/4361/wiki/results/figure-7-pure/figure-7-pure-16.png"><img src="https://static.igem.wiki/teams/4361/wiki/results/figure-7-pure/figure-7-pure-16.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. 2: A67H mutant.</figcaption>
 
</figure>
 
</figure>
  

Latest revision as of 18:23, 13 October 2022


BlcR A67H

A mutant of the BlcR protein, created through site-directed mutagenesis with primers R3 (Part:BBa_K4361218) and F3.6 A67H (Part:BBa_K4361224). For this mutant, the alanine in position 67 has been changed to histidine by mutating the GCG codon to CAT.

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

  • A 478 > T, resulting in substitution T160S
  • Insertion of A after A 732, resulting in a possible frameshift and extension of the protein
  • Insertion of A after A 800, 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 16), production in an E. coli based cell free system was not successful, no bands corresponding to BlcR were visible.
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: A67H mutant.