Part:BBa_K781001

[R0010][B0034] - RFP-FliC Insertion Chimera

This is the modified RFP protein obtained from J04450, that has been incorporated into the E. coli flagellin as an insertion.

This part can be used as a template for making other PCR overlap insertions. After transforming the product, and digesting the template plasmid with Dpn1, any template that has not been successfully digested will form red colonies.

This part was successfully characterized and determined to show a lower expression of red fluorescence compared against the positive control J04500. This result can be expected for a number of possible reasons.

• The chimeric protein may not be as stable as the free RFP.
• The RFP may be polymerizing in flagella, resulting in a quenching effect as the fluorescent light may be absorbed by other RFP molecules arranged in close proximity.
• The flagellin domains may hinder proper folding of the RFP. This may be why the more constrained deletion variant is showing less expression compared to the insertion variant, which has more flexibility.

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BamHI site found at 2173
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal AgeI site found at 530
  Illegal AgeI site found at 938
  Illegal AgeI site found at 1552
  Illegal AgeI site found at 1664
  
• 1000
  COMPATIBLE WITH RFC[1000]