Coding

Part:BBa_K3140008

Designed by: Fahad Ali   Group: iGEM19_Sydney_Australia   (2019-10-12)


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[1]. Mutation of the highly-conserved LOV domain cystine residue (Cys73) to alanine will convert VVD into a fluoroprotein. In addition, previous work [2] 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.

We conducted a fluorescence assay using a TECAN Spark microplate reader Escherichia coli TOP10 cultures containing VVD36-C73A, VVD36-C73A-CH1, VVD36-C73A-CH2, VVD36-C73A-CH3, VVD36-C73A-CH4, and VVD36-C73A-CH4-M130I in pK18 (Fig. 2). As shown, VVD36-C73A-CH4-M130I exhibits the greatest fluorescence, followed by VVD36-C73A-CH2. All other VVD38-C73A variants were only slightly more fluorescent than control.

Fig. 2: Graph of TECAN OD-normalised fluorescence for Escherichia coli TOP10 cultures containing VVD36-C73A, VVD36-C73A-CH1, VVD36-C73A-CH2, VVD36-C73A-CH3, VVD36-C73A-CH4, and VVD36-C73A-CH4-M130I in pK18.

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]


  1. Schwerdtfeger, C. & Linden, H. VIVID is a flavoprotein and serves as a fungal blue light photoreceptor for photoadaptation. EMBO J 22, 4846-55 (2003).
  2. Zoltowski, B.D. et al. Conformational switching in the fungal light sensor Vivid. Science 316, 1054-7 (2007).
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