A comprehensive summary of all the most important results and achievements by the UCopenhagen iGEM team 2019 is listed here.

Biosensor Results

General workflow

Figure1: Throughout the project we used the same general workflow to create our yeast strains: PCR amplification → USER ligation → E. coli cloning → E. coli colony PCR → Sequencing → Plasmid purification → Yeast transformation → Yeast colony PCR

Design

Our ambition was to create a chewing gum, containing a yeast biosensor, which can detect sex hormones in saliva and elicit a quantifiable response based on the levels of these. In our wet lab work, we decided to focus on creating two different biosensors with receptors for estrogen and Luteinizing Hormone (LH) respectively.

The two receptors we are using are the G-Protein-Coupled Estrogen Receptor (GPER), and the Xenopus Laevis Lutropin-Choriogonadotropic Hormone Receptor (XLHCGR), to detect estrogen and LH respectively. You can read more about the receptors and our general design on our Project Design page.

A minimal biosensor requires five modules (Fig. 2A). To construct our minimal biosensors, we chose to take advantage of the yeast pheromone response pathway. To do this, we engineered our yeast strains to include one of our receptors (GPER or XLHCGR respectively), a chimeric adaptor module, connecting the receptor to the yeast signaling pathway, and a reporter module eliciting a fluorescent response. For our signal processing module, we chose to converse the endogenous MAP kinase signaling cascade normally used in the pheromone response pathway (Fig. 2B).

Figure 2: The conserved and engineered modules of our minimal biosensor | 2A: The required modules of a minimal biosensor. 2B: The MAP Kinase signaling pathway already present in yeast was conserved, while the sensing module was engineered to fit a hormone receptor. The adaptor module was engineered to connect the receptor to the signaling pathway, and the reporter module was engineered to a fluorescent signal response.

Multiplex cassette systems

To integrate our biosensor system into the yeast genome, we used a multiplex 5-assembler genomic integration cassette (5-modular system), which allowed for integration into the yeast chromosome 10, site 3 (Fig. 3A). In order to confirm successful expression and localization of our receptors, we also utilized a multiplex 3-assembler genomic integration cassette (3- modular system) (Fig. 3B) to construct yeast strains containing our receptors linked to superfolder GFP (sfGFP). See Figure 6 for a complete explanation of yeast strains created.

Figure 3: The multiplex genomic integration system | 3A: The 5-assembler genomic integration cassette. The first module contains the receptor. Second module contains the GPA1. Third assembler contains the transcription factor STE12. Fourth assembler contains an empty vector backbone and the fifth assembler contains the reporter gene ZsGreen. 3B: The 3-assembler genomic integration cassette. The first module contains the receptor tagged with sfGFP. The second and the third assemblers contain empty backbone vectors.

Vector construction

For construction of all our yeast strains we used the same general workflow. Initially all fragments were PCR amplified using primers carrying USER overhangs. The fragments were verified through gel electrophoresis (Fig. 4A). Following PCR amplification, the fragments, were purified and ligated into our integration vectors using USER cloning. For a detailed explanation of the backbones used, please refer to our Project Design page. See Table 1 for list of constructs.

Table 1: The constructs made by USER ligation

Backbone	Promoter	Gene fragment
X3A	pCCW12	GPER
X3A	pCCW12	XLHCGR
X3A	pCCW12	GPER-linker + sfGFP
X3A	pCCW12	XLHCGR-linker + sfGFP
Ass2A	pPGK1	GPA1-Gαi
Ass2A	pPGK1	GPA1-Gαs
Ass2B	pRET2	STE12
X3C	pFIG1	ZsGreen

Following USER cloning, successful transformant were picked and colony PCR was performed. The colony PCR products were separated by Gel electrophoresis and positive colonies were determined by comparing the band sizes on the gel with expected sizes. Figure 4B depicts a representative colony PCR gel electrophoresis, as performed on GPER-Li-sfGFP. Based on the results of colony PCR, 1-2 colonies were picked and grown overnight. Plasmid DNA purification was carried out and sequencing used to verify successful vector construction.

Figure 4: Representative gel electrophoresis images | 4A: PCR of USER fragments. Expected band sizes are listed for each fragment. 4B: Colony PCR of ligated fragment pCCW12-GPER. A positive band was observed in lane 2 around 1800 bp, which correlates well to the expected band size for a construct of 1818 bp. Lane 1 does not show a band, which means that this sample was negative.

Yeast transformation and colony PCR

The purified vectors were transformed into S. cerevisiae, creating a total of 5 different strains (Fig. 5). Three strains were made using the 3-assembler cassette (OV1, OV2, OV3), and two using the 5-assembler cassette (OV4, OV5).

Figure 5: Overview of the 5 different cassettes transformed into yeast | OV1 is a negative control strain transformed with empty vector backbones. OV2 and OV3 were transformed with our 2 receptors conjugated to sfGFP in module 1, and two empty vectors in module 2 and 3. OV4 and OV5 were transformed with our receptors in module 1, the adaptor GPA1 in module 2, and the transcription factor STE12 in module 3. Module 4 was kept empty in these strains, while module 5 contains the reporter ZsGreen.

Correct integration was verified using PCR. In the presence of our construct, we expect to see a band at 1000 bp, and in the absence of the constructs, we expect to see the bands at 1500 bp. For an in depth explanation of detecting positive yeast colonies, please refer to our Project Design page.

Figure 6 depicts gel electrophoresis from yeast colony PCR of the 2 strains (OV2 and OV3) transformed with our sfGFP tagged receptors. In both gel images, bands of 1000 bp were observed, which confirmed that both the receptors tagged with sfGFP have been successfully integrated into the yeast genome.

Figure 6: OV2 and OV3 yeast colony PCR gel images | Specific yeast genotyping primers were used for the PCR reaction. PCR products were separated by electrophoresis on 1% agarose gel. The sizes of the molecular weight standards are shown on the left. Lanes 1-8 represent individual colonies. 6A: Bands of the colony PCR product of yeast expressing GPER-Li-sfGFP (OV2). 6B: Bands of the colony PCR product of yeast expressing XLHCGR-sfGFP (OV3). In both A and B, we see bands around 1 kb, confirming positively transformed yeast.

Figure 7 depicts gel electrophoresis from yeast colony PCR of the 2 strains (OV4 and OV5) transformed with our full minimal biosensor systems. In both gel images, bands of 1000 bp were observed, which confirmed that both minimal biosensor systems containing receptors for estrogen and LH respectively, have been successfully integrated into the yeast genome.

Figure 7: OV4 and OV5 yeast colony PCR gel images | Specific yeast genotyping primers were used for the PCR reaction. PCR products were separated by electrophoresis on 1% agarose gel. The sizes of the molecular weight standards are shown on the left. Lanes 1-8 represent individual colonies. 7A: Bands of the colony PCR product of yeast expressing the biosensor system containing GPER (OV4). 7B: Bands of the colony PCR product of yeast expressing the biosensor system containing XLHCGR (OV5). In both A and B, we see bands around 1 kb, confirming positively transformed yeast.

Confirmation of receptor expression and localization
To confirm the correct expression and localization of the GPER and XLHCGR in our biosensor yeast strains (OV4 and OV5), we utilized the strains transformed with sfGFP tagged receptors (OV2 and OV3). Using these strains, we carried out western blotting with antibodies against GFP. The resulting gel images confirmed that our yeast strains were expressing the receptors (Fig. 8, Fig 9). Strain OV1 transformed with empty vector backbones was used as a negative control, and as expected showed no expression of sfGFP. A yeast strain constitutively expressing sfGFP was used as a positive control.

Figure 8: Western blot of yeast strain OV2 (GPER-Li-sfGFP) with anti-GFP | 8A: OV2, soluble fraction. Clear bands are seen for both tested samples, as well as for the positive control. 8B: OV2, insoluble fraction. Faint bands are seen for both tested samples, as well as for the positive control. Strain OV1 was used as a negative control and showed no expression of sfGFP.

Since the sfGFP is conjugated to the C-terminus of the receptor, GFP expression confirms the receptor expression. The protein size of the receptor is 44 kDa while the protein size of sfGFP is 27 kDa. Thus, we would expect to see a band of 71 kDa. However, we observed bands around 32 kDa. This might indicate that the receptor is expressed in a truncated form. We observed that most of the receptor was present in the insoluble fraction. This result indicates that either the receptor is located on the plasma membrane, as the insoluble parts include all membrane-bound proteins, or that the receptor, folds poorly, thus forming inclusion bodies.

Figure 9: Western blot of yeast strain OV3 (XLHCGR-sfGFP) with anti-GFP | 9A: OV3, soluble fraction. Clear bands are seen for both tested samples, as well as for the positive control. 9B: OV3 insoluble fraction. Faint bands are seen for both tested samples, as well as for the positive control. Strain OV1 was used as a negative control and showed no expression of sfGFP.

Expected band sizes are of 107 kDa. XLHCGR-Li-sfGFP was found in the soluble fraction, indicating its presence within the cytosol and not anchored to the plasma membrane as expected. Also, the band size around 32 kDa indicates that the receptor might have been expressed in a truncated form.

To further examine receptor expression, confocal microscopy was performed on strains OV2 and OV3 (expressing GPER-Li-sfGFP and XLHCGR-sfGFP, respectively). Results were compared to the negative control strain OV1 (Fig. 10 and Fig. 11).

Figure 10: Confocal microscopy of strains OV1 and OV2 | 10A and 10B: Strain OV2 expressing GPER-sfGFP, fluorescence and bright field, respectively. 10C and 10D: Positive control strain, constitutively expressing sfGFP, fluorescence and bright field, respectively 10E and 10F: Strain OV1 expressing empty vectors, fluorescence and bright field, respectively.

The confocal images above further confirmed the expression of the GPER conjugated to sfGFP in strain OV2. Furthermore, they confirmed the proper alignment of the receptor, as sfGFP is tagged to the C-terminus of the receptors, which is expressed inside the cell. When comparing the images of the OV2 strain to the positive control, it is evident that the receptor is localized to the plasma membrane, as we see a concentric ring in the yeast cells. As expected, no fluorescence was observed in the negative control strain OV1.

Figure 11: Confocal microscopy of strains OV2 and OV3 | 11A and 11B: Strain OV3 expressing XLHCGR-sfGFP, fluorescence and bright field, respectively. 11C and 11D: Positive control strain, constitutively expressing sfGFP, fluorescence and bright field, respectively 11E and 11F: Strain OV1 expressing empty vectors, fluorescence and bright field, respectively.

The confocal images further confirmed the expression of the XLHCGR conjugated to sfGFP in strain OV3. Furthermore, they confirmed the proper alignment of the receptor, as sfGFP is tagged to the C-terminus of the receptors, which is expressed inside the cell. However, from the images, intracellular localization of the XLHCGR could not be confirmed. As expected, no fluorescence was observed in the negative control strain OV1.

Bioactivity assay: GPER biosensor

In order to test the activity of our GPER biosensor, OV4, we carried out a bioactivity assay with strain OV4. Here, OV4 cells were induced with increasing concentrations of estradiol (estrogen), where after the fluorescent intensity was measured and compared to that of our control strain, OV1 (Fig. 12).

Figure 12: Fluorescent bioactivity assay of OV4 strain | The blue line indicates the fluorescence (RFU) from the negative control strain OV1 induced with increasing concentrations of estradiol. The orange line indicates the fluorescence (RFU) from the OV4 strain containing the minimal biosensor with GPER induced with increasing concentrations of estradiol. The Y-axis indicates RFU, while the X-axis indicates estradiol concentrations in picomolar.

As seen in Figure 12, no significant difference in fluorescence could be detected between the biosensor (OV4) and the negative control (OV1) when induced with estradiol. This indicated that the biosensor did not work as intended.

Bioactivity assay: Nuclear estrogen receptor

To further investigate the potential of a functional yeast estrogen biosensor, we ordered the Plasmid #69100 (pRS416-yZ3EV-Z3pr-yEGFP (RB3579)) from the Addgene catalog. This vector carries the transcription factor Z3EV which is activated by estrogen and induces yEGFP expression from the same plasmid. For more information on the receptor, please refer to our Project Design page.

Estrogen in the nucleus should lead to an increased fluorescence compared to a control. Using the same general workflow as described above, we transformed the receptor into yeast, creating strain OV(NR). To test the functionality of the yeast strain, we set up a bioactivity assay identical to the one that was performed on strain OV4.

Figure 13: Fluorescent bioactivity assay of strain OV(NR)| The blue lines indicates the fluorescence (RFU) from yeast transformed with empty vector, induced with increasing concentrations of estradiol. The orange line indicates the fluorescence (RFU) from the OV(NR) strain containing the Addgene Plasmid #69100, when induced with increasing concentrations of estradiol. The Y axis indicates RFU, while the X axis indicates estradiol concentrations in picomolar.

As seen in Figure 13, no significant difference in fluorescence could be detected between the biosensor (OV(NR)) and the negative control strain, containing an empty vector, when induced with estradiol. This indicated that the biosensor with the nuclear estrogen receptor did not work as intended, either. A possible source of error is insufficient estrogen concentration in the nucleus, or crystal formation of estrogen, as described below.

Estrogen crystal formation

To get a better understanding, we chose to examine our induced strain OV4 under the fluorescent microscope (Fig. 14). Here, we found that the estradiol formed crystals which elicit a fluorescent response. As such, these crystals might have masked any significant fluorescent responses of the cells. An option to further test the biosensors would be to perform flow cytometry, which would measure the fluorescence of each individual cell or crystal fragment. Through this method we should be able to distinguish cells from crystals, and thus filter out the crystals from the measurements. As such, we would be able to compare the fluorescent signal of cells induced with different concentrations of estradiol. However, we were not able to carry out these experiments within the timeframe of the iGEM competition.

Unfortunately, we were unable to get a hold of LH, the necessary agonist to test the functionality of the XLHCGR biosensor, expressed in strain OV5. Thus, these experiments will have to be carried out at a different time.

Figure 14: Fluorescence microscope image of estrogen crystal formation | 14A and 14B: Strain OV4, fluorescence and bright field, respectively. Here we see the estradiol forming crystals.

Conclusions

We successfully constructed five yeast strains using a multiplex genomic integration system. Through multiple methods, we demonstrated that the receptors are expressed, and that at least one of them is likely to locate to the plasma membrane. We can also conclude that there is a chance the receptors might be truncated, or is processed in another way, and that they might form inclusion bodies. We set up an assay to test the functionality of our biosensors, and we can conclude that the estradiol forms crystals, which interfere with the assay.

Kill Switch Results

To meet the individual consumer’s and the general public’s concern, it is important that the genetically modified yeast we are using is not able to survive outside its designated environment. To account for this, we decided that our yeast should contain a kill switch. Here, we chose to utilize the BAX-BI-1 system, which is a toxin-antitoxin system, as an initial proof of concept. Here, BAX works as a toxin inducing cell death, while BAX inhibitor-1 (BI-1) serves as an anti-toxin inhibiting the effect of BAX (Figure 15).

Function of BAX

The way BAX induces cell death has been extensively studied and is well summarized by Westphal et al. (2011)¹: To put it briefly, BAX naturally takes part in the intrinsic apoptosis pathway in higher eukaryotes. When the cell experiences stress, BAX is activated to oligomerize in the outer mitochondrial membrane to form pores. These enable cytochrome c to exit into the cytoplasm where it will activate caspases, important effectors of a controlled intrinsic cell death. The yeast genome doesn’t contain any homologues of the mammalian BAX protein; this raises the question of whether yeast responds to BAX in a similar way as to what mammalian cells do. Previous studies have shown that BAX expression in yeast indeed leads to cell death even though the pathway for its activation is not present. Furthermore, yeast has been proven to be a suitable vehicle for analysis of Bcl-2 proteins, the protein family that BAX is part of³.

Function of BI-1

The BAX inhibitor BI-1 can generally be regarded as promotor of cell survival. It locates to the membrane of the ER and inhibits the activation of BAX and the following pro-apoptotic effects. Besides this, BI-1 prevents cell death through BAX independent mechanisms and takes a critical role in the Ca2+ homeostasis².

Kill Switch Design

We constructed a kill switch by placing a toxin under a constitutively active promoter and the antitoxin under an inducible promoter, where the inducer is only present in the media. The yeast strain used should contain BI-1 under the control of an inducible promoter and BAX under the control of a constitutive promoter. The inducer should then be present in the designated environment; in our case the gum. As such, if the yeast escapes the gum, and therefore becomes separated from the inducer, apoptosis is induced and the yeast dies. For more information on the kill switch device, please refer to our Project Design page. With this in mind, we took inspiration from the work of the 2017 iGEM team, NAU_China 2017, and decided to set up two sets of experiments.

Figure 15: Overview of the BAX/BI-1 Kill switch device | The figure depicts the genes encoding for the toxin BAX under the expression of a constitutive promoter while the anti-toxin BI-1 under the expression of an inducible promoter pGAL1. In the absence of galactose, BI-1 will not be expressed, and the toxin will be allowed to kill the yeast cell.

Characterization

Firstly, we decided to characterize the biobrick submitted by NAU_China 2017 (BBa_K2365048, a BAX mutant), by expressing it in yeast under the control of the galactose inducible promoter, pGAL1. Here, we wanted to evaluate the efficiency of BAX as a yeast toxin by investigating how different concentrations of galactose in the growth media can induce cell death.

Improvement

Secondly, we decided to improve the BI-1 biobrick (BBa_K2365518), also submitted by NAU_China 2017, by also placing it under the control of the GAL1 promoter. By placing BI-1 under the control of the pGAL1 transcription should only occur in the presence of galactose. We saw this improvement as necessary in order to contain the yeast in a defined environment, as a yeast strain that expresses BI-1 under control of the GAL1 promoter and BAX constitutively, will only be able to survive if enough galactose is present. If a yeast cell escapes its defined environment, the supply of this sugar will likely drop, BI-1 expression is interrupted, which will shift the toxin-antitoxin equilibrium in favour of BAX.

As we imagined the modules for the biosensor and the kill switch to be incorporated into the same yeast strain, we chose a different site for the genomic integration for the kill switch sequences, namely chromosome XI site 2.

Vector construction

As with our biosensor, all fragments were PCR amplified with primers containing USER overhangs and sizes confirmed using gel electrophoresis. After purification of the fragments, we used USER cloning to create the constructs listed in table 2. All constructs were verified using sequencing.

Table 2: Constructs made by USER ligation.

Backbone		Promoter	Gene Fragment
1	XI2A	pGAL1	BAX
2	XI2A	pADH2	BAX
3	XI2A	pTEF1	BAX
4	B(Ass2)	pGAL1	BI-1
5	XI2C	-	-
6	pUUS	pTEF1	BAX
7	pUUS	pADH2	BAX
8	pUUS	pGAL1	BAX
9	pUUS	-	-
10	pWUS	pGAL1	BI-1
11	pWUS	pTEF1	BI-1
12	pWUS	-	-

Yeast transformation and colony PCR

As part of this study, a total of four strains (OV1, OV6 to OV8; Figure 16) were created using the 3-assembler system listed above (Figure 3), with insertion into chromosome 11. For all transformations yeast colony PCR was used to confirm correct chromosomal integration (Figure 17).

Figure 16: Overview of the 3 different cassettes transformed into yeast.

Figure 17: Yeast colony PCR of the transformed constructs. Specific yeast genotyping primers were used for the PCR reaction. We expected bands around 900 bp for positive colonies and 1500 bp for untransformed colonies. Unfortunately, there were no positive colonies for the pADH2-BAX+pGAL1-BI-1 construct (not shown).

Galactose induction assay

To analyse the effect of BAX on our yeast under different expression levels, we conducted a galactose induction assay using raf-U plates with five different galactose concentrations. Cultures of yeast containing pGAL1-BAX (OV6) or empty vector (OV1) were grown O/N and then diluted to an OD_600nm of 0.5. On plates with either 0%, 0.025%, 0.05%, 0.1% or 0.2% galactose, 10 µl of each culture were spotted in increasing dilutions (10-1 to 10-4; Figure 18 and 19). After three days of incubation at 30 °C, two observations were made. First of, the strain with the integrated pGAL1-BAX construct showed decreased growth compared to the control strain even when galactose was absent (Figure 18). This suggests that the galactose promoter is leaky and a low amount of BAX is produced at all times.

Figure 18: Growth of OV1 and OV6 in the absence of galactose. The cultures were spotted in the dilutions 10-1 to 10-4 of an OD_600nm of 0.5 and incubated for three days at 30 °C.

Secondly, when comparing the spots of OV6 at a dilution of 10^-1 at different galactose concentrations, a clear inverse correlation of CFU/ml and percentage of galactose in the media can be observed. This suggests that successful induction of BAX leads to apoptosis in our yeast.

Figure 19: Growth comparison of OV6 and control strain OV1 in the presence of varying galactose concentrations. Shown are the CFUs at an OD of 0.05 after incubation at 30 °C for four days. The three yellow colonies seen at 0.2% galactose in OV6 can be morphologically distinguished from the others, suggesting that they are contaminants.

Quantitative galactose induction assay of pGAL1-BAX

To further analyze the effect of BAX on our yeast under different expression levels, we conducted a quantitative galactose induction assay. Here, an O/N culture of yeast containing pGAL1-BAX (OV6) was diluted to an OD_600nm of 0.5. Subsequently, 100 µl of the culture in dilutions of 10^-3 and 10^-4 were spread on plates with 0%, 0.05%, 0.1%, 0.3% and 1% galactose, respectively. Each plate was made in duplicates. After incubation for three days at 30 °C, the CFU/ml were calculated and compared to the control (Figure 20).

Figure 20: Gradient induction of BAX in yeast using the inducible GAL1 promoter. Shown are the CFUs per ml that have resulted from cultures with an OD_600nm of 0.5 in presence of varying galactose concentrations (0%, 0.05%, 0.1%, 0.3% and 1%). If no errorbar is indicated, only one of the used duplets showed a quantifiable amount of colonies.

As seen on figure 20, a clear reduction in growth was seen already at low galactose concentrations (0.05 %). In addition it appears BAX was expressed in sufficient concentrations to kill the yeast cells at a galactose concentrations of 0.3 %. Unfortunately, our control strain (OV1) was only plated on raf-U with 1 % galactose. However, no reduction in growth was seen at this concentration (compared to OV6 grown in 0 % galactose media).

In addition to a reduction in the number of colonies formed between the two strains, there was also a clear difference on the size of the colonies. Only very small colonies were formed upon induction of BAX, this indicates that the cells were killed before a normal sized colony could be formed. To test this, the OV6 plate was incubated for an additional day at 30 °C and subsequently left at room temperature. No additional growth was observed (Figure 21).

Figure 21: Comparison of colony sizes of OV1 and OV6 grown on raf-U agar with galactose. Left: Colonies of the control strain OV1 (dilution 10-3 ) in the presence of 1% galactose after incubation for three days at 30 °C. Right: Colonies of the pGAL1-BAX strain OV6 (dilution 10-3) in the presence of 0.1% galactose after incubation for four days at 30 °C.

Dual Plasmid transformation

In addition to the strains above, an additional set of strains were constructed using yeast expression plasmids (Figure 22).

Figure 22: Schematic overview of the plasmids the strains OV9-18 were transformed with.

Following transformation, the yeast cultures were plated. However, the transformed strains only grew partly as expected and later we encountered problems cultivating them in liquid media, which is why we decided against carrying out further experiments with them.

BAX rescue assay

We wanted to examine whether BI-1 is able to neutralize the lethal impact of BAX, and therefor performed a rescue assay. In this, both BAX and BI-1 were put under control of the GAL1 promoter (OV19).

For construction of the strain, we transformed the OV6 strain (pGAL1-BAX integrated into the genome) with a high-copy number pGAL1-BI-1 plasmid.

For the assay colonies were picked and diluted in sterile water. Using the comparative galactose induction assay as a template, the ODs at 600 nm were equalised to 0.035 and different dilutions of the colonies (10-0 to 10-3) were spotted on plates with Glu-UW agar and raf-U-W agar with 1% galactose in volumes of 10 µl. A yeast strain containing only pGAL1-BAX and the empty pWUS plasmid served as control (OV20) and was spotted along with the sample (Figure 23).

Figure 23: BAX rescue assay. In this assay, the strains OV19 and OV20 were grown on both glu-U-W agar (left) and raf-U-W (1% galactose) agar in different dilutions of OD_600nm(10^-0 to 10-3).

As BAX was expressed under the inducible promoter pGAL1 in both strains, we expected normal growth on the glu-U-W plates. On the raf-U-W plates with 1% galactose, we expected the control strain OV20 to show a decreased colony size and number compared the strain OV19 that should be rescued by the BI-1 plasmid. However, as seen in figure 23, no significant growth was observed for either strain in the presence of galactose. As such, we saw no indication that BI-1 was able to prevent BAX induced apoptosis. While it is possible that the lack of function of BI-1 could be the result of a loss of function during transformation, our data suggests that BI-1 is not able to prevent BAX induced apoptosis in our system, and therefore is not suitable for our kill switch.

References

1. Westphal, D., Dewson, G., Czabotar, P. E., Kluck, R. M. (2011): Molecular biology of BAX and Bak activation and action. Biochimica et biophysica acta 1813 (4), pp. 521–531.
2. Li, B., Yadav, R. K., Jeong, G. S., Kim, H-R., Chae, H-J. (2014): The characteristics of BAX inhibitor-1 and its related diseases. Current molecular medicine 14 (5), pp. 603–615.
3. Polčic, P., Jaká, P., Mentel, M. (2015): Yeast as a tool for studying proteins of the Bcl-2 family. Microbial cell (Graz, Austria) 2 (3), pp. 74–87.