Team:EPFL/Engineering

Espress'EAU - Engineering Success

Synthetic Biology

Reporter strains

Our aim is to build a yeast strain that generates a signal in the presence of pesticides in water. In order to accomplish this, we leverage the stress response pathway of yeast. We hypothesize that the latter should be activated by the pesticides, as yeast has previously been shown to be sensitive towards pesticides1,2,3. By combining the promoter region of stress induced genes with a reporter gene, we expect the reporter to be activated, when the yeast is grown in contaminated water, and thus, generate a signal. Here, we will describe the strategy we used to successfully synthesize these reporter strains. For more results obtained with these strains, please visit Reporter Strains.

After studying the stress response of yeast, we selected five genes, which are known to be activated when the yeast cell is exposed to stress: HSP124, TRX25, GLR16, YCF17, GSH18. We therefore decided to engineer five yeast reporter strains, one for each stress response gene.

This engineering process comprises two main steps:

  1. Assembling the cassette that we want to integrate into the yeast genome

  2. Cassette integration (yeast transformation)

Cassette assembly

As mentioned above, we want to integrate a promoter and a reporter gene into the yeast genome. However, other parts are also required in order to optimize the integration and expression of the reporter. Multiple parts can be assembled together simultaneously using the Golden Gate assembly method. But, as the number of parts that are being assembled increases, the probability of them assembling in the correct order and directionality decreases. Our initial cassette design consisted of 8 separate parts that are based on the yeast toolkit described by Lee et al. 20159.


Figure 1

Figure 1: The eight part types of our cassette design.


After running the assembly experiment several times and not obtaining the desired results, we decided to modify our experimental design. Instead of trying to assemble the 8 parts at once, we decided to start by assembling 6 parts and then add the remaining 2 parts in a second assembly step. By decomposing this process into two assembly steps, we effectively reduce the number of parts concurrently coming together, and thus increase the probability of a successful assembly. By following this alternative method, we obtained our desired cassettes.

Cloning strategy

The next step is to integrate the cassettes into the yeast genome. An efficient way to achieve this is by using CRISPR/Cas910. By transforming the Cas9 backbone along with the sgRNA and the linearized integration cassette into the yeast cells, the Cas9-sgRNA complex will be synthesized and create a double strand break at the desired locus. The yeast’s homology repair (HR) mechanism then repairs this break while introducing the given cassette at the break site.


Figure 2

Figure 2: The linearized sgRNA has 500bp exact homology arms that are used to gap-repair with the Cas9 backbone in yeast, thereby forming a stable expression vector. Cas9 will then induce double strand breaks in the yeast genome.

Results and Discussion

To verify our cassette assembly, we transform the plasmids into E.coli to amplify them and perform a colony PCR. When running the PCR products on a gel (Figure 3), we observe bands at the expected sizes indicating that our five integration cassettes were assembled successfully. The cassettes were then sequenced for further validation.

Figure 3

Figure 3: Colony PCR of E.coli colonies containing our cassettes after the first assembly step (6-part assembly). The correct cassette should yield a band with a size of 2470bp while the backbone should yield a band with a length of 1612bp. The screening process yielded several colonies that produced bands of the correct size (highlighted in green) for each of the five cassettes that we constructed.


To confirm the successful integration of the cassette, we do a selection step against the selection marker (URA3) which is found in the Cas9 backbone, and perform a colony PCR on the transformed yeast colonies. In Figure 4 we observe bands at the expected sizes for four of our strains indicating that the cassettes successfully integrated the yeast genome. The data for the GSH1 cassette integration is missing.

Figure 4

Figure 4: Colony PCR of four yeast strains transformed with the final integration cassette. The primers used for this were located in the Lys2 homology arm region that was added during the second assembly. Bands of the correct sizes (highlighted in green) can be observed for each strain.


These strains were then sequenced for further validation. The following sequencing results showcase the successful integration of the cassettes into the yeast genome.


Figure 5: Sequencing alignments for the four transformed yeast strains. The forward and reverse sequencing primers are aligned with the yeast genome. We can see that the promoters along with the reporter genes and the terminators have been integrated into the yeast genome.


Hardware

When imagining our project, we passed through several different hardware ideas. First, we planned to have a continuous monitoring device for water quality based on a bioreactor. Then, after different discussions with our stakeholders which challenged our ideas, we thought that it would be more useful to have a punctual measurement and adapted the project to the need of non-trained users. For this, a small device was conceived to enable the users to measure yeast growth along with a fluorescent signal in case of a stressing environment.

To test our components, first a calibration curve was also done using our optical density detector (based on a light-to-frequency converter) and demonstrated a linear relation between the frequency that was recorded by our detector and on a commercial spectrophotometer. This shows that the device that our Hardware Team built was successful and is functional to record yeast growth using the capsules that we designed.


Figure 2

Figure 6: Linear relation between the OD600 measured with 11 standard samples in a spectrophotometer (x axis) and the transmitted light measured by our device (y axis).


The fluorescence sensor was also tested. As we aimed to detect a red fluorescent signal (expressed by mScarlet-I protein), the fluorescence at different wavelengths (green, red and blue) was measured. An inducible strain that can be activated by -estradiol was used as a positive control as it gives a red fluorescent signal when activated. The graph below shows a remarkable increase in the detected signal when measuring this inducible strain compared to the negative control (water) and compared to the blue and green wavelength.

Figure 2

Figure 7: Fluorescence measurements with inducible strain for 3 different wavelengths.


Even though none of the components are very complex, we consider the design and implementation of the device an engineering success: the prototype built runs smoothly for multiple hours (16+) with no issues, it is very compact, and every component works well in parallel. The user feedback that we got the 23rd and 27th of October was as well very positive showing that the device is simple to use. In addition to being functional, it is close to be usable by the interested users.



References

  1. Braconi D, Bernardini G, Santucci A. Saccharomyces cerevisiae as a model in ecotoxicological studies: A post-genomics perspective. Journal of proteomics. 2016 Mar 30;137:19-34.

  2. Lal S, Lal R. Bioaccumulation, metabolism, and effects of DDT, fenitrothion, and chlorpyrifos onSaccharomyces cerevisiae. Archives of environmental contamination and toxicology. 1987 Nov 1;16(6):753-7.

  3. Esteve K, Poupot C, Dabert P, Mietton-Peuchot M, Milisic V. A Saccharomyces cerevisiae-based bioassay for assessing pesticide toxicity. Journal of industrial microbiology & biotechnology. 2009 Dec 1;36(12):1529-34.

  4. Varela, J. C., Praekelt, U. M., Meacock, P. A., Planta, R. J. & Mager, W. H. The Saccharomyces cerevisiae HSP12 gene is activated by the high-osmolarity glycerol pathway and negatively regulated by protein kinase A. Molecular and Cellular Biology 15, 6232–6245 (1995).

  5. Kuge, S. & Jones, N. YAP1 dependent activation of TRX2 is essential for the response of Saccharomyces cerevisiae to oxidative stress by hydroperoxides. The EMBO Journal 13, 655–664 (1994).

  6. Grant, C. M., Collinson, L. P., Roe, J. H. & Dawes, I. W. Yeast glutathione reductase is required for protection against oxidative stress and is a target gene for yAP-1 transcriptional regulation. Mol. Microbiol. 21, 171–179 (1996).

  7. Bhuiyan, M. S. U. et al. Overexpression of a yeast cadmium factor 1 (YCF1) enhances heavy metal tolerance and accumulation in Brassica juncea. Plant Cell Tiss Organ Cult 105, 85–91 (2011).

  8. Wu, A. L. & Moye-Rowley, W. S. GSH1, which encodes gamma-glutamylcysteine synthetase, is a target gene for yAP-1 transcriptional regulation. Molecular and Cellular Biology 14, 5832–5839 (1994).

  9. Lee ME, DeLoache WC, Cervantes B, Dueber JE. A highly characterized yeast toolkit for modular, multipart assembly. ACS synthetic biology. 2015 Sep 18;4(9):975-86.

  10. DiCarlo JE, Norville JE, Mali P, Rios X, Aach J, Church GM. Genome engineering in Saccharomyces cerevisiae using CRISPR-Cas systems. Nucleic acids research. 2013 Apr 1;41(7):4336-43.

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