Part:BBa_K4361315

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 (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 16), it was concluded that the production was not successful due to the bands corresponding to BlcR being too poorly distinguishable.

Figure 1. SDS PAGE gel of PURE reactions with GreenLys and a variant of Part:BBa_K4361106 containing a BlcR mutant. Arrows indicate bands corresponding to BlcR dimers and monomers, respectively. C = negative control, WT = wildtype BlcR.