Difference between revisions of "Part:BBa K4361319"

Revision as of 18:10, 13 October 2022

BlcR L38V

A mutant of the BlcR protein, created through site-directed mutagenesis with primers R1 (Part:BBa_K4361200) and F1.3 L38V (Part:BBa_K4361203). For this mutant, the leucine in position 38 has been changed to valine by mutating the TTG codon to GTG.

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

G 9 > T, resulting in substitution Q3H
G 148 > T, resulting in a possible stop codon in position 50
G 156 > C, silent mutation
CGCG 198-201 > ATAT, resulting in substitution A67Y
A 364 > T, resulting in substitution T122S

Sequencing data only contained this part's sequence up until and including nucleotide 399. 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

Experimental results

The set of BlcR mutants (Part:BBa_K4361300 through this part) 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 the protein was produced. For this mutant (lane 20), 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. 19: L38V mutant.

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 <html>
 <h3>Experimental results</h3>
-The set of BlcR mutants (</html>[[Part:BBa_K4361300]] through this part<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  the protein was produced. For this mutant (<u>lane 20</u>), a band was visible on the gel at the expected position around ~35 kDa. This confirms that the mutant can be produced in an <i> E. coli </i> based cell-free system.
+The set of BlcR mutants (</html>[[Part:BBa_K4361300]] through this part<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  the protein was produced. For this mutant (<u>lane 20</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-20.png"><img src="https://static.igem.wiki/teams/4361/wiki/results/figure-7-pure/figure-7-pure-20.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. 19: L38V mutant.</figcaption>
 </figure>