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[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).
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.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]
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