Part:BBa_K4238004

C120-trucCYC1p+mCherry+ADH1t
When transformed into yeast together with BBa_K4238003, this part causes the yeast to produce red fluorescence under blue light. The dimeric EL222 binds to the C120 sequence in the C120-trucCYC1promoter under blue light, allowing transcription of the downstream sequence. The expression can be quantified with the fluorescence of mCherry. A similar part was created in which a kill switch (BBa_K4238005) deriving from the human Bax gene is used instead of mCherry.

Biology

EL222, which consists of a light-oxygen-voltage-sensing (LOV) domain and a DNA binding domain, helix-turn-helix (HTH) domain, is connected to an activation domain (AD), VP16. In the dark, this complex does not bind to DNA because the LOV domain masks the HTH domain. When blue light (450 nm) hits them, the structure of the LOV domain changes, freeing the HTH domain, which binds to each other to form a homodimer that binds to the C120 region in the DNA. The VP16 domain then promotes the expression of the downstream gene [1][2].

Wet lab result

Using this part as a reporter gene, we tested this parts BBa_K4238003in plasmid and genome.

Plasmid:

Figure 1. The completed plasmid

The created sequences were transformed into yeast and assayed. First, the yeast was incubated in the dark for at least 24 hours to reset the photoreceptor and other components. After that, we separated the strains into two groups. The former strain was incubated under blue light with an intensity of 347[μW/cm^2] using the Optocoder, and fluorescence was observed using a microscope 24 hours after the start of the illumination (blue). The latter strain was observed under a microscope after incubation in the dark (dark). Observations were conducted at two conditions: transmitted light (exposure time: 50 ms) and TxR (exposure time: 200 ms). The results are shown in figure 2 and 3 below.

Figure 2. Distribution of fluorescence. 349 cells of blue and 441 of dark were observed.

Figure 3. Average of fluorescence. Error bars indicate standard errors. * denotes a significant difference at p<0.05, ** denotes a significant difference at p<0.01, and n.s. denotes no significant difference. The statistical test was conducted using the Wilcoxon rank sum test.

The expression of mCherry was lower when illuminated with blue light, which contradicts the expectation. After we consulted professors, we found out that this might be because the plasmids were lost from the yeast since they were cultured in non-selective medium.

Genome Integration:

Figure 4. The completed plasmid

The homologous sequences were added upstream and downstream of the EL222 sequence and introduced into the locus YNRCΔ9 on the yeast genome. We generated the sequence of C120-trucCYC1promoter-mCherry-CYC1terminator as a reporter for EL222 by OE-PCR, and homologously recombined it to the genomic locus YMRWΔ15.

The created sequences were transformed into yeast and assayed. First, the yeast was incubated in the dark for at least 24 hours to reset the photoreceptor and other components. After that, we separated the strains into two groups. The former strain was incubated under blue light with an intensity of 420[μW/cm^2] using the Optocoder, and fluorescence was observed using a microscope 3 hours (3h) and 6 hours (6h) after the start of the incubation. The latter strain was observed under a microscope after incubation in the dark(dark). Observations were made at two wavelengths: transmitted light (exposure time: 50 ms) and TxR (exposure time: 200 ms). The results are shown in Figure 5 below.

Figure 5. Average of fluorescence. Error bars indicate standard errors. * denotes a significant difference at p<0.05, ** denotes a significant difference at p<0.01, and n.s. denotes no significant difference. The statistical test was conducted using the Wilcoxon rank sum test.

After 3 hours of blue light irradiation, gene expression was about 3 times higher than that without irradiation, confirming that EL222 was functioning properly. On the other hand, the irradiation for 6 hours did not change the gene expression from the 3-hour time point. This indicates that gene expression had already peaked at 3 hours after irradiation, and that 3 hours of light exposure was sufficient to activate the blue light-dependent promoter.

Reference

[1] Motta-Mena, L. B., Reade, A., Mallory, M. J., Glantz, S., Weiner, O. D., Lynch, K. W., & Gardner, K. H. (2014). An optogenetic gene expression system with rapid activation and deactivation kinetics. Nature chemical biology, 10(3), 196-202. doi: 10.1038/nchembio.1430
[2] "Team:NUS Singapore - 2021.igem.org." https://2021.igem.org/Team:NUS_Singapore (accessed Oct. 09, 2022)

Sequence and Features

Assembly Compatibility:

• 10
  INCOMPATIBLE WITH RFC[10]

  Illegal XbaI site found at 104
  Illegal SpeI site found at 79
  
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal NheI site found at 86
  Illegal SpeI site found at 79
  
• 21
  COMPATIBLE WITH RFC[21]
• 23
  INCOMPATIBLE WITH RFC[23]

  Illegal XbaI site found at 104
  Illegal SpeI site found at 79
  
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal XbaI site found at 104
  Illegal SpeI site found at 79
  
• 1000
  COMPATIBLE WITH RFC[1000]