Part:BBa_K4361300

BlcR D37R

A mutant of the BlcR protein, created through site-directed mutagenesis with primers R1 (Part:BBa_K4361200) and F1.1 D37R (Part:BBa_K4361201). For this mutant, the aspartic acid in position 37 has been changed to arginine by mutating the GAC codon to CGC.

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

• G 763 > A, resulting in substitution E255K

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 designed to be used in a PURE reaction together with Part:BBa_K4361115 and Part:BBa_K4361117 to translate the mutants and subsequently measure their binding strength to the wildtype Blc operator and a mutant, respectively. As the results of these experiments were inconsistent, it was concluded that PURE was not the correct system to screen BlcR activity. However, through the addition of GreenLys (fluorescent Cy2-labeled lysine), the results from PURE could still be used to show whether or not the mutants were produced in solution (see Figure 1.). For this mutant (lane 1), it was concluded that the production was not succesful 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.