Team:Sydney Australia/Improve



The Original Part

The original part that we have improved this year is a Vivid protein from the fungi, Neurospora crassa , found in the iGEM registry as part:BBa_K1616014. This protein is a member of the Light-Oxygen-Voltage (LOV) protein domain. LOV domain proteins are blue light sensors and are conserved in fungi, plants and prokaryotes (Müller and Weber, 2013). When a single amino acid change is made in the Vivid protein, converting a cysteine at residue to an alanine, it become a weakly fluorescent (Chapman et al., 2008). We were able to perform error prone PCR on this weakly fluorescent protein to make a strongly fluorescent protein that has many advantages over existing fluorescent proteins such as GFP.

Improving the Part

Firstly, the vivid gene sequence from the part BBa_K1616014 page was codon harmonised for more effective expression in E. coli . Then, 36 amino acids were removed from the end of protein. This truncation does not change the photochemical properties of the protein; it simply makes the protein more stable and soluble at room temperature (Zoltowski et al., 2007). Lastly, the amino acid at residue 73 was changed from a cysteine to an alanine to make the protein weakly fluorescent.

This gene was ordered as a G-Block and cloned into E. coli. PCR was used to amplify the gene with taq polymerase. MnCl2was added to a concentration of 15mM to increase the error rate of the polymerase. The resulting PCR products were column purified, digested with restriction enzymes, and then ligated into plasmids. These plasmids were transformed into E. coli and plated on agar plates. After being allowed to grow for 48 hours, the colonies were examined under long-wave UV light. Several colonies that were more brightly fluorescent than the rest were identified (see Figure 1).

Figure 1: Results from initial ligation and transformation of error-prone PCR products into TOP10 cells. Extra bright colonies are indicated with yellow arrows.

In total, 12 bright colonies were picked and re-streaked. The vivid gene in each of the colonies was amplified with PCR and sent off for sanger sequencing. The sequencing results were then aligned with the original codon harmonised fluorescent vivid gene, to identify if there were conserved changes across the mutants. It was found that there was a conserved mutation at the methionine at residue 130. This methionine was changed to either isoleucine, which was found in 5 of the mutants, tryptophan, which was found in 3 of the mutants, or leucine, which was found in one of the mutants (see Figure 2).

Figure 2: Sequence alignment of error-prone PCR mutants with original VVD gene, with the region of common M130 mutations circled.

A G-Block was ordered that had the same sequence as the initial fluorescent vivid gene but contained the most commonly conserved mutation in the brighter colonies - the methionine at residue 130 was replaced with an isoleucine. The new vivid gene was then expressed in E. coli and plated alongside E. coli containing the original fluorescent vivid genes. This allowed us to determine that it definitely was the mutation of the methionine residue that caused the increased fluorescence (see Figure 3).

Figure 3: The Met130Ile vivid mutant compared to the weakly fluorescence wild type vivid and 4 codon harmonised vivid variants (CH1, CH2, CH3 and CH4) under long wave UV light.

A fluorescence assay was performed that compared the original codon harmonised fluorescence Vivid, the wild type fluorescent Vivid, an empty plasmid, and the new brighter Met130Ile Vivid protein (see Figure 4). This shows the extent to which the single amino acid mutation increased the fluorescence of the protein.

Figure 4: Graph of TECAN fluorescence data from the VVD-Met301 g-block cells and the original VVD_wt and VVD_CH4 cells.
The new and improved fluorescent protein has been uploaded to the parts registry as part:BBa_K3140010.

Uses and advantages of this new fluorescent protein

This new part has several advantages over other fluorescent proteins such as GFP. Firstly, its small size (150 amino acids) means that it will be less disruptive when used as an intracellular tag. Secondly, it does not require oxygen to be fluorescent, unlike GFP and derivatives (Mukherjee et al., 2013).

Based on results from other homologous LOV-domain derived fluoroproteins, this new fluorescent protein is also likely to be stable at a wider pH and temperature range than GFP, and will mature fluorescence more rapidly (Mukherjee et al., 2013). Further characterisation is required to fully assess its properties.

Uses and advantages of this new fluorescent protein

Through an amino acid substitution at the M130 position (Figure 2) brighter Vivid fluorescence proteins can be generated. Error-prone PCR can be a high throughput method for finding novel mutations and novel phenotypes.


  1. Chapman, S., Faulkner, C., Kaiserli, E., Garcia-Mata, C., Savenkov, E., Roberts, A., Oparka, K. and Christie, J. (2008). The photoreversible fluorescent protein iLOV outperforms GFP as a reporter of plant virus infection. Proceedings of the National Academy of Sciences, 105(50), pp.20038-20043.
  2. Zoltowski, B., Schwerdtfeger, C., Widom, J., Loros, J., Bilwes, A., Dunlap, J. and Crane, B. (2007). Conformational Switching in the Fungal Light Sensor Vivid. Science, 316(5827), pp.1054-1057.
  3. Müller, K. and Weber, W. (2013). Optogenetic tools for mammalian systems. Molecular BioSystems, 9(4), p.596.
  4. Mukherjee, A., Walker, J., Weyant, K. and Schroeder, C. (2013). Characterization of Flavin-Based Fluorescent Proteins: An Emerging Class of Fluorescent Reporters. PLoS ONE, 8(5), p.e64753.