Difference between revisions of "Part:BBa K4361314"

Latest revision as of 15:57, 12 October 2022

BlcR A67V

A mutant of the BlcR protein, created through site-directed mutagenesis with primers R3 (Part:BBa_K4361218) and F3.5 A67V (Part:BBa_K4361223). For this mutant, the alanine in position 67 has been changed to valine by mutating the GCG codon to GTG.

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

G 482 > T, resulting in substitution G161V
G 670 > T, resulting in substitution A224S
G 677 > T and T 678 > G, resulting in substitution R226L
G 716 > C, resulting in substitution S239T
G 765 > A, silent mutation
G 787 > C, resulting in substitution A263P

Sequencing data only contained this part's sequence up until and including nucleotide 791. All nucleotides after are presumed to be identical to those found in the original codon optimized BlcR, Part:BBa_K4361100.

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 15), 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. 15: A67V mutant.

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 <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 15</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 15</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>
 <a href="https://static.igem.wiki/teams/4361/wiki/results/figure-7-pure/figure-7-pure-15.png"><img src="https://static.igem.wiki/teams/4361/wiki/results/figure-7-pure/figure-7-pure-15.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. 15: A67V mutant.</figcaption>
 </figure>