Part:BBa_K3140008

VVD36-C73A-CH3 (relative proportion harmonisation)

VVD36-C73A-CH3 is a fluoroprotein derived from the VIVID (VVD) blue-light photoreceptor in Neurospora crassa. It is a codon harmonised variant of VVD36-C73A, making use of our relative proportion harmonisation method.

Usage and Biology

VIVID (VVD) is a blue-light sensing photoreceptor from the ascomycete (spore-shooting fungus) N. crassa. It is a member of a family of proteins containing a light-oxygen-voltage-sensing (LOV) domain, which modulate circadian responses to environmental stimuli. Mutation of the highly-conserved LOV domain cystine residue (Cys73) to alanine will convert VVD into a fluoroprotein. In addition, previous work indicates that truncation of the first 36 amino acids of VVD increases its stability in heterologous systems. Our VVD part incorporates both of these changes.

VVD36-C73A-CH3 is a 456 bp sequence, encoding a 151 amino acid peptide (17.2 kDa). We cloned VVD36-C73A, VVD36-C73A-CH1, VVD36-C73A-CH2, VVD36-C73A-CH3, and VVD36-C73A-CH4 into pK18, which were then transformed into Escherichia coli TOP10 cells. In order to validate the insertion of these genes, we conducted a PCR with primers that were specific to the pK18 backbone, which should a 624 bp product. This was verified with agarose gel electrophoresis, demonstrating had the expected size (Fig. 1).

Fig. 1: Agarose gel electrophoresis of VVD36-C73A, VVD36-C73A-CH1, VVD36-C73A-CH2, VVD36-C73A-CH3, VVD36-C73A-CH4, and VVD36-C73A-CH4-M130I conducted on 1% agarose gel in 1X TAE for 50 min at 100 V.

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  COMPATIBLE WITH RFC[1000]