Overview

In our project, Optopass, we aimed to develop the brand new safety and security system for S. cerevisiae. This system consists of three key parts: light inducible promoter system, site-specific recombination system, and kill-switch. We performed seven experiments to test these elements, gathering the deep understanding and Proof of Concept.

0-1 Constitutive Promoters

Design

The first step we took was to test different types of constitutive promoters and figure out the character of each promoter. Last year, the UTokyo team used the CYC1 promoter as a constitutive promoter, but its expression level could be dependent on oxygen concentration. We tested ADH1 promoter and TDH3 promoter as alternatives. The sequences we created are shown in Figure 1 below.

**Figure 1**. Completed plasmids of each promoter.

Build

Fluorescent protein mCherry was placed at the downstream of each promoter and terminated by CYC1 terminator. These inserts were connected with vector pRS316 by Seamless Ligation Cloning Extract (SLiCE), and then transformed into E. coli JM109 competent cells. After the successful transformation to JM 109, completed plasmids were extracted, and then transformed into S.cerevisiae BY4741.

**Figure 2**. Photographs of yeast colonies.

**Figure 3**. Photographs of electrophoresis. All sequences were confirmed.

Test

Yeast transformed with plasmids containing each of the three promoters were observed under a microscope and the mCherry fluorescence was compared. These yeast were picked up from colonies and inoculated on YPD medium. Images were taken at two conditions: transmitted light (exposure time 50 ms) and TxR (exposure time 200 ms). Results are shown in Figures 4 and 5 below.

**Figure 4**. Distribution of fluorescence. 156 cells of ADH1, 166 of CYC1, and 168 of TDH3 were measured.

**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.

Learn

In the case of all three promoters, many cells were emitting almost no fluorescence. This is probably because the cells were cultured in a non-selective medium and some of them lost their plasmids. On average, the ADH1 promoter had about the same expression level as the CYC1 promoter, while the TDH3 promoter was almost 20-fold higher. Too high expression is not appropriate for Optopass, which uses proteins like kill switches and recombinases, which can work even at the very low concentration. Therefore, we decided to use the ADH1 promoter for our gene circuits.

0-2 Improvement of ADH1 promoter

We conducted the parts improvement of the existing ADH1 promoter BBa_K2365039, both to increase the expression level and to make it easier to handle. We cut the first 209 bp of the original 409 bp ADH1 promoter (which we name ADH1p long) to obtain 409 bp ADH1p short. We connected each of them with the fluorescence gene mCherry, transformed yeasts with the plasmids, and measured the fluorescence. As a result, newly created ADH1p short proved to be more effective in protein expression. See our BBa_K4238000.

1 EL222

Light

Design 1

The first step in Optopass is to receive the light input. As a promoter reacting to blue light, we assayed the EL222 system. The sequences we created are shown in the Figure 6 below. Constitutively expressed EL222 proteins will receive blue light and dimerize, which makes it possible to bind to the C120 region near the trucCYC1 promoter. Then the VP16 domain in the EL222 protein activates the transcription of downstream mCherry, fluorescent protein. In short, yeast transformed with this plasmid are supposed to express mCherry when blue light is shone on them. See our Description page to find more about EL222.

Build 1

The EL222-VP16 complex and the promoter sequence (BBa_K3570021) were ordered from IDT. This sequence was introduced into S. cerevisiae by homologous recombination together with pRS316 vector.

**Figure 7**. Photographs of electrophoresis. All sequences were confirmed.

Test 1

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 $347 [\mathrm{\mu W/cm^2}]$ 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 8 and 9 below.

**Figure 8**. Distribution of fluorescence. 349 cells of blue and 441 of dark were observed.

**Figure 9**. 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.

Learn 1

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.

Design 2

In order not to let the yeast lose the transformed sequences, we decided to integrate the sequences into the yeast genome, rather than transform them as plasmids. Since the whole sequence was too long to integrate into the single locus, we separated it into two: EL222 and reporter. The sequences we created are shown in Figure 10 below.

Build 2

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.

**Figure 11**. Photographs of yeast colonies.

**Figure 12**. Photographs of electrophoresis. All sequences were confirmed.

Test 2

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 $420 [\mathrm{\mu W/cm^2}]$ 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 13 below.

**Figure 13**. 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.

Learn 2

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.

EL222 worked successfully!

2 synTALE & zinc finger

Light

Design

In Optopass, the light-dependent promoters form the first part, input. We had planned to use synTALE as the DNA-binding protein for the red light-dependent promoter system, but discussions with our partner Chalmers-Gothenburg's team raised the possibility of using zinc finger. Experiments were conducted to see which was more suitable for our project. See Description page to learn more about synTALE and zinc finger.

Build

First, synTALE and zinc finger sequences were designed. The two 9-base target sequences used by Chalmers-Gothenburg were linked together to form an 18-base target sequence, and synTALE and zinc finger that bind to this sequence were designed. synTALE protein consists of 34-amino acid repeat modules, each recognizing one base [1]. Therefore, the amino acid sequences of the domains that recognize each of 4 bases (A, G, C, T) and the N- and C-terminal domains were determined based on [2] and [3], and these were connected to make the overall amino acid sequence. It was then codon-optimized and synthesized by a private company. Each domain of zinc finger protein recognizes 3 bases. The amino acid sequence of 9 bp recognizing zinc finger used by Chalmers-Gothenburg was adjusted based on [4], and a linker connecting the two zinc finger was designed based on [5]. It was then codon-optimized and synthesized by a private company as well.

The 18-nucleotide sequence recognized by synTALE and zinc finger was inserted into the TDH3 promoter so that transcription would be repressed when synTALE and zinc finger bind to it. According to [6], placing the recognition sequence slightly behind the TATA box is effective for transcriptional repression, so we identified the position of the TATA box of the TDH3 promoter by referring to [7] and inserted the recognition sequence behind the TATA box. mCherry was placed downstream of the promoter. We named the complete block "reporter."

The completed sequence is shown in Figure 14 and 15 below. reporter was generated in vitro by OE-PCR and transformed into the genome of S. cerevisiae BY4741. synTALE and zinc finger were transformed as plasmids by homologous recombination together with fragments of pRS316.

**Figure 14**. The completed sequences for the reporter.

**Figure 15**. The completed sequences for synTALE and zinc finger.

**Figure 16**. Photographs of yeast colonies.

Test

The expression of mCherry, the reporter, in three yeast strains were compared by microscopic fluorescence observation: one in which only the reporter was transformed and neither synTALE nor zinc finger was present (control), one in which a plasmid of synTALE was transformed in addition to the reporter (synTALE), and one in which a plasmid of zinc finger was transformed in addition to the reporter (zinc finger). The samples used for microscopic observation were inoculated on YPD medium from colonies for the control, and inoculated on SC-U medium from colonies for the synTALE and zinc finger, respectively, and observed with transmitted light (exposure time: 50 ms) and TxR (exposure time: 200 ms). The results are shown in Figure 17 and Figure 18 below.

**Figure 17**. Distribution of fluorescence. 313 cells of control, 210 of synTALE, and 170 of zinc finger were observed.

**Figure 18**. 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.

Learn

In synTALE, the fluorescence of mCherry was significantly lower than that of control, suggesting that synTALE bound to the target sequence and inhibited the transcription of the gene. On the other hand, no significant difference was observed for zinc finger compared to control, indicating that zinc finger does not bind to the target sequence and cause transcriptional inhibition. This suggests that synTALE is superior to zinc finger in terms of the binding to DNA. Therefore, we decided to use synTALE instead of zinc finger for the red-light-sensor in Optopass.

synTALE proved to be stronger than zinc finger

3 Cre-loxP

Order

Design

In Optopass, light inputs are received as sequences. In order to "memorize" the previous stimuli, we use site-specific recombination system. We assayed one example of recombination system: Cre-loxP system. In this system, when Cre is expressed, it cuts the gene between two loxP sites irreversibly. See our Description page to find more about the recombination system. The sequences we created are shown in Figure 19 and 20 below. In Figure 19, which indicates the test strain, Cre will cut mCherry and CYC1 terminator between two loxP sites and mNeonGreen, which will then be placed direct under the ADH1 promoter, will be expressed. However, in Figure 20, which indicates the control strain, Cre will not be expressed and thus mCherry rather than mNeonGreen will be transcripted.

**Figure 19**. The completed sequences for the test.

**Figure 20**. The completed sequences for the control.

Build

We transformed two modules in S. cerevisiae BY4741. First one is for the reporter system. Under constitutive ADH1 promoter, mCherry and CYC1 terminator was placed between two loxP sites, followed by mNeonGreen, a green fluorescent protein, and ADH1 terminator. This module supposed to expresses mCherry before recombination and mNeonGreen after recombination. This module was genome integrated to the YMRWΔ15 locus. Strain which only has this module 1 was used as control.

Second module consists of ADH1 promoter, Cre and CYC1 terminator. This insert was converted with vector pRS316 by SLiCE, then it was transformed to E. coli JM109 competent cell. After the successful transformation to JM 109, target plasmid was extracted. We added the plasmid to the control strain and used it as a test strain.

**Figure 21**. Photographs of yeast colonies.

**Figure 22**. Photographs of electrophoresis. All sequences were confirmed.

Test

The fluorescence of mCherry and mNeonGreen in yeast transformed with only the loxP sequence (control) and yeast transformed with Cre in addition to the loxP sequence (test) were compared by microscopic observation. The samples used for microscopic observation were inoculated on YPD medium in the case of control and on SC-U medium in the case of test, and were observed with setting of transmitted light (exposure time 50 ms), TxR (wavelength 560 nm, exposure time 500 ms), and GFP (wavelength 470 nm, exposure time 1000 ms). The results are shown in Figures 23-25 below.

**Figure 24**. Distribution of mCherry fluorescence brightness for control and test.

**Figure 25**. Distribution of mNeonGreen fluorescence brightness for control and test.

Learn

The scatter plots show that the fluorescence brightness of both mCherry and mNeonGreen decreased when cells were transformed with Cre. However, when looking at the distribution of each fluorescence protein, mCherry was originally expressed by most cells and stopped being expressed when Cre was transformed, while mNeonGreen was originally not expressed by most cells and that did not change even after Cre was transformed. This suggests that recombination was successfully performed and mCherry was cut out, but mNeonGreen was not expressed for some reason. This might be because an extra sequence between the promoter and mNeonGreen affected the expression level, or mNeonGreen did not work in S. cerevisiae because it derives from S. pombe. If this inference is correct, then we have confirmed that the recombination of Cre and loxP proceeds satisfactorily under the conditions of this experiment.

Cre-loxP probably worked successfully!

4 Light-Inducible Recombination

Light Order

Design

After it was confirmed that recombination occurs sufficiently, the next step was to combine this recombination system with blue light-dependent recombination system. Light-dependent recombination is an essential part of Optopass. It is important to construct a blue-light dependent recombination system and investigate the relationship between the time of light exposure and recombination possibility. The sequences we created are shown in Figure 26 below. When yeast are cultured in the dark, Cre does not work and mCherry will be expressed. When cultured under the blue light, however, EL222 activates the expression of Cre, which cuts mCherry and CYC1 terminator, resulting in the expression of mNeonGreen.

Build

The three sequences shown in Figure 26 were homologously recombined into yeast genome. Leu-deficient medium was newly prepared as the selection medium. OE-PCR was used to design the sequences as in previous experiments. loxP-containing sequences were purchased from IDT, Leu2 was extracted by PCR from pRS313, and mNeonGreen was shared with us from the S. pombe strain in our laboratory.

**Figure 27**. A photograph of yeast colonies.

Test

The prepared gene sequences were transformed into yeast while they were kept in dark, then the fluorescence of mCherry and mNeonGreen was observed under a microscope after they were treated in three ways: without light (dark), with blue light for 3 hours continuously (3h), and with blue light for 6 hours continuously (6h). The blue light was irradiated using an Optocoder with an intensity of $420 [\mathrm{\mu W/cm^2}]$ . The samples used for microscopic observation were inoculated on YPD medium from colonies. We observed them under the light of three wavelengths: transmitted light (exposure time 50 ms), Texas Red (exposure time 500 ms), and GFP-B (exposure time 1000 ms). Figures 28 and 29 below display the results.

**Figure 28**. Scatter plots of mCherry and mNeonGreen brightness values of 3h, 6h, and dark. 202 cells of 3h, 196 of 6h, and 114 of dark were observed.

**Figure 29**. Enlarged view of Figure 28.

Learn

The distribution of fluorescence was not much different when exposed to blue light for 3 hours or 6 hours than when not exposed to light. There are two possible reasons for this: (1) recombination did not occur even with light exposure, or (2) recombination occurred even without light exposure. To confirm which of the two is correct, we compared the results with control (3_control) and test (3_test) of the previous experiment 3 Cre-loxP. 3_control are the cells in which recombination has not occurred, and 3_test are the cells in which recombination has occurred. In the third experiment, mNeonGreen was found not to be expressed after recombination, so only mCherry expression was compared. Figure 30 below shows the result.

**Figure 30**. 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 luminance of light-inducible recombination cells is significantly lower than that of 3_control and 3_test cells. Since 3_control cells are before recombination and 3_test cells are after recombination, it can be concluded that recombination has occurred in all dark, 3h, and 6h, i.e., recombination has progressed even without light exposure. This is consistent with the conclusion in the modeling that the threshold for recombination is so low that recombination can proceed easily. See the Modeling page for details. Therefore, the system needs to be improved by raising the threshold in the same manner as discussed in the modeling.

Light-inducible recombinase worked, but the leakage was too large.

5 Light-Inducible Kill Switch

Light KillSwitch

Design

Kill switch is essential to safety and security. We constructed a light-inducible kill switch and assayed it. When this kill switch works, it can be applied to biosecurity at the citizen level, since yeast equipped with this kill switch will kill itself when it gets out into the environment and is exposed to sunlight. The sequences we created are shown in Figure 31 below. When the yeast receives blue light, EL222 activates the expression of human Bax gene (hBax), which has been proved to induce apoptosis. See our Description page to find more about kill switch.

Build

Since we could not succeed in genome integration before wiki freeze, we decided to transfer the hBax gene by plasmid.

The plasmid was constructed using SLiCE(Seamless Ligation Cloning Extract) method. The method is based on homologous recombination in E. coli. It is possible to link linear sequences with homologous sequences.The completed plasmid was transfected into E. coli (dh5a competent cells), amplified, and transformed into yeast with the EL222 sequence homologously recombined into the genome.

**Figure 32**. A photograph of yeast colonies.

Test

Yeast were transformed with completed sequences, inoculated into SC-U medium and exposed to blue light continuously and cell density was measured over time (kill light). As a control, cell counts were also measured when the cells were grown in the dark without light (kill dark). In addition, to investigate the possibility that light itself is damaging the growth of yeast, we cultured BY4741, an untransformed strain, in YPD, and measured the cell counts in light (BY4741 light) and dark (BY4741 dark) conditions. Measurements were taken every 30 minutes until 4 hours after light exposure, and again 2 days later. Blue light with an intensity of $420 [\mathrm{\mu W/cm^2}]$ was irradiated using an Optocoder. The results are shown in Figures 33 and 34 below.

**Figure 33**. Time course of cell concentration under each condition, expressed as a relative value with the concentration at 30 min as 1.

**Figure 34**. Enlarged view of Figure 33.

Learn

The cell density of BY4741 cells exposed to light was lower than that of the cells not exposed to light after 2 days, suggesting that exposure to light may reduce the proliferation rate of the cells. The number of cells of the kill switch-transfected strains did not differ significantly for the first 4 hours, but after 2 days, the number of cells in the light-transfected group was slightly lower than that in the non-light-transfected group. However, it could not be determined whether this difference was due to the kill switch or because light exposure suppressed the proliferation rate.

Light-inducible kill switch didn't work.

Future Prospect

About the light inducible promoter system

Blue light (EL222)

We will try to improve the system by increasing the number of C120 repeats from 1 to 5.

Red light (PhiRex)

Using synTALE, we will assay the PhiRex system to see if it works.

Other wavelengths

We will construct promoter systems reacting to other wavelengths to make the cryptogram more complicated
We will see if different promoters interfere with each other or not.

About the site specific recombination system

Using other fluorescent proteins instead of mNeonGreen, we will assay the system again to see if it really works.
We will assay PA-Cre to see if the threshold is increased, as was shown in the modelling.

About the kill switch

We will assay the kill switch again, extending the time length to track the cell count.

About the combination of basic elements

We will combine the basic elements to create the whole system of Optopass.

References

[1] Morbitzer, R., Elsaesser, J., Hausner, J., & Lahaye, T. (2011). Assembly of custom TALE-type DNA binding domains by modular cloning. Nucleic acids research, 39(13), 5790-5799. doi: 10.1093/nar/gkr151

[2] Sanjana, N. E., Cong, L., Zhou, Y., Cunniff, M. M., Feng, G., & Zhang, F. (2012). A transcription activator-like effector toolbox for genome engineering. Nature protocols, 7(1), 171-192. doi: 10.1038/nprot.2011.431

[3] Hochrein, L., Machens, F., Messerschmidt, K., & Mueller-Roeber, B. (2017). PhiReX: a programmable and red light-regulated protein expression switch for yeast. Nucleic acids research, 45(15), 9193-9205. doi: 10.1093/nar/gkx610

[4] Beerli, R. R., Segal, D. J., Dreier, B., & Barbas III, C. F. (1998). Toward controlling gene expression at will: specific regulation of the erbB-2/HER-2 promoter by using polydactyl zinc finger proteins constructed from modular building blocks. Proceedings of the National Academy of Sciences, 95(25), 14628-14633. doi: 10.1073/pnas.95.25.14628

[5] Kim, J. S., & Pabo, C. O. (1998). Getting a handhold on DNA: design of poly-zinc finger proteins with femtomolar dissociation constants. Proceedings of the National Academy of Sciences, 95(6), 2812-2817. doi: 10.1073/pnas.95.6.2812

[6] Kim, J. S., & Pabo, C. O. (1997). Transcriptional repression by zinc finger peptides: exploring the potential for applications in gene therapy. Journal of Biological Chemistry, 272(47), 29795-29800. doi: 10.1074/jbc.272.47.29795

[7] Wedler, H., & Wambutt, R. (1995). A temperature-sensitive lambda cl repressor functions on a modified operator in yeast cells by masking the TATA element. Molecular and General Genetics MGG, 248(4), 499-505. doi: 10.1007/BF02191651