Team:EPFL/Reporter Strains

Espress'EAU - Reporter Strains

Abstract

Saccharomyces Cerevisiae is a model organism being able to defend itself against various adverse environmental conditions with its inherited stress response pathway. We hijacked the native yeast stress response pathway and created several reporter strains that can respond to various environmental contaminants such as pesticides and other chemical compounds. We placed the regulated promoters (HSP12pr, TRX2pr, YCF1pr, GSH1pr, GLR1pr) upstream of a reporter protein, i.e. mScarlet-I, which will be turned on by certain transcription factors (YAP1, MSN2, MSN4) in the stress response pathway.

Design

Promoters

We selected the 500bp upstream of the open reading frame of a selection of genes that are upregulated upon stress response. The length of 500bp was chosen to make sure that all the important transcription factor binding sites would be included.

Reporter

We chose mScarlet-I as the fluorescence reporter, which has an excitation wavelength of 569 nm and an emission wavelength of 593 nm. We placed the promoter sequences upstream of the coding sequence of mScarlet-I and the sequence was codon optimized for expression in S. Cerevisiae using Benchling.

Terminator

The strong terminator tADH1 was used for all of the five cassettes.

Figure 1

Figure 1: The transcriptional unit consists of a 500bp long promoter region followed by the coding sequence for mScarlet-I that was codon optimized for the expression in yeast using Benchling. The same terminator tADH1 from the Yeast Toolkit 2 was used for all the five cassettes.

gBlock design

To create a part plasmid that is compatible with the Yeast Toolkit, we designed gBlocks consisting of the 500bp promoter region that was retrieved from the yeast genome database 3 and added overhangs to both ends. These linkers contained a BsaI and a BsmBI cutting site each to make Golden Gate assembly possible.

Cloning

To construct the five integration cassettes we used parts that we created ourselves and combined them with parts from the Dueber Yeast toolkit (YTK)4. An exhaustive list of all the parts that were used or created can be found on our parts page.

Construction of part plasmids

The gBlocks were inserted into the part plasmid entry vector (pYTK001) to create part plasmids through a BsmBI assembly. This step was performed by our teaching assistant Shiyu Cheng.

Assembly of transcriptional units

The transcriptional units were assembled with a BsaI assembly using an ampicillin selection marker backbone. The parts that are used to do this assembly contain 1. the left connector (pYTK002) 2. the promoter (BBa_K3600000-BBa_K3600005) 3. the reporter gene (BBa_K36000XX) 4. the terminator (pYTK053) 5. the right connector (pYTK072) and 6. the backbone (pYTK095). This assembly yields the composite parts (BBa_K36000XX-BBa_K36000XX).



Assembly of final cassette

The final cassette was assembled by a BsmBI assembly from two parts: 1. the transcriptional unit (BBa_K36000XX-BBa_K36000XX) and 2. the Lys2-GFP dropout multigene backbone (BBa_K36000XX).

Figure 2: The five cassettes that were assembled differ only in the promoter region. KanR was used as a selection marker for E.Coli.

Results

Figure 3

Figure 3: A GFP dropout backbone from the yeast toolkit that carries an ampicillin resistance gene (pYTK095)[3] was used in this assembly step to allow for both positive and negative selection on an agar plate treated with ampicillin. Under blue light it becomes apparent which of the colonies carry only the backbone (green) and which ones carry a plasmid that lost the GFP coding sequence (white).

Figure 4

Figure 4: The colonies that successfully dropped the GFP sequence in the first assembly step were screened with colony PCR (cPCR). YTK AmpR validation primers were used. Their binding sites are located in the ColE1 and in the AmpR sequence of the backbone plasmid (pYTK095)[3]. 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 for each of the five cassettes that we constructed.

Yeast Tranformation

Method

The method we used is based on an article published by W.Shaw from the Ellis lab5. It relies on the capability of the Cas9 protein to induce double-strand breaks (DSB) in specific locations. S. Cerevisiae has a preference for homologous recombination (HR) over non-homologous end joining (NHEJ). This mechanism is exploited by transforming the yeast with our assembled cassette and a Cas9-sgRNA expression vector at the same time. The cassette contains homology arms for the Lys2 locus which is targeted by the sgRNA.

Figure 5

Figure 5: The Cas9-sgRNA expression vector carries the coding sequence of Cas9 under the strong Pgk1 promoter as well as the selection marker URA3. By digesting it with BsmBI it is linearized and can be gel purified. The sgRNA was linearized with an EcoRV and the cassette with a NotI digestion. (adapted from a schematic figure from LBNC unpublished data)

Figure 6

Figure 6: 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. (adapted from a schematic figure from LBNC unpublished data)

Figure 7

Figure 7: After transformation, yeast was plated on SC-URA plates so only cells that contained the URA3 selection marker which is found on the Cas9-sgRNA vector would survive. (adapted from a schematic figure from LBNC unpublished data)

Figure 8

Figure 8: To cure the transformed strains from the sgRNA-Cas9 expression vector they were transferred to 5-FOA plates. Cells that still contain the URA3 marker will produce Fluorouracil (5-FU) which will kill these cells. (adapted from a schematic figure from LBNC unpublished data)

Results

Figure 9

Figure 9: The cassettes containing the designed transcriptional unit were digested with NotI (1-5). The cassettes have all the same size and differ only in their promoter sequence. This digestion is expected to yield bands with a length of 2955 and 1858bp. The sgRNAs were digested with EcoRV (6,7) and the bands are expected to have a size of 1022 and 1880bp. The Cas9-sgRNA expression vector is expected to yield bands at 10050 and 1030bp. The higher band is then gel extracted and purified. On this gel, the purified linearized Cas9-URA3 backbone is shown (8).

Figure 10

Figure 10: After yeast transformation the colonies were once again screened with cPCR. The primers used for this were located in the Lys2 homology arm region that was added during the second assembly. For the cassette with the GSH1 promoter we did not find any strain that was successfully transformed after two iterations.

Figure 11: 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.

Fluorescence Microscopy

Method

We used a fluorescence microscope to characterize the mScarlet-I expression. Overnight cultures were grown in yeast extract peptone dextrose (YPD) medium and suspended in synthetic complete (SC) medium after washing with water. SC medium is used for the final suspension as it is less autofluorescence than YPD to reduce the background signal. Atrazine with a concentration of 1mg/L was added to the cells. The pesticide was dissolved in methanol and the final methanol concentration was 1%.

Results

Figure 12

Figure 12: As a control experiment, wildtype yeast was observed. In the bright-field image (left) it can be seen that some of the cells are most probably dead. Some of the dead cells have a rather high signal (red circle) on the fluorescence image (right). The fluorescence signal of living cells was quantified using imageJ. The mean signal (n=15) was found to be at 12.1928 after correcting for the background.

Figure 12a Figure 12b Figure 12c Figure 12d

Figure 13: 1mg/L Atrazine in methanol was added to overnight cultures to test if the expression of mScarletI could be induced. The final concentration of methanol is 1%. The signal was quantified using imageJ. The fluorescence signals were corrected for the background.

The data we collected shows that for all the four strains that were tested, the fluorescence signal is higher than for the wildtype control. We conclude from this that the promoters work and that the reporter protein mScarlet-I can be expressed by our strains. A difference between the cells that were exposed to 1mg/L atrazine and the control is observed. The t-test at 5% was significant for three out of the four strains (HSP12, GLR1 and YCF1). However, it is not clear if this difference is due to the pesticide (atrazine) or the solvent (methanol). A better controlled experiment will have to be conducted to conclude about this. The high signal in the cells without pesticide and methanol could be due to the stress response being triggered due to starvation.

Pesticide Assay

Method

Finally, to assess the influence of pesticides on our reporter strains, we performed fluorescence measurements for more than 20 hours in presence of pesticides. As before, overnight cultures were grown in YPD and suspended in SC medium after washing with water. We added Atrazine as well as Metolachlor (concentration of 1 mg/L dissolved in Methanol) and compared the different reporter strains with or without pesticides, as well as with a Methanol solution (as a control) against wild-type. 4 replicates for each measurement have been performed.

Results

Figure 13

Figure 14: Normalized fluorescence (mScarlet-I) by OD600 measurements across time for HSP12 reporter strain (4 replicates) and wild-type in presence of pesticides and a Methanol solution 1% (control). Curves were filtered with a short time Fourrier transform (sampling frequency of 100). Times below 5 hours has been discarded (too low OD600, incoherent normalization).

As we can see from Figure 14, the HSP12 strain in presence of Metolachlor (1 mg/l) produces a significantly higher fluorescent signal than a wild-type strain (without Metolachlor, with or in presence of Methanol only). Additionally, adding pesticides to the HSP12 strain makes the cells more fluorescent than an HSP12 strain alone or in presence of the solution of Methanol (1%). One of the replicates reaches a peak after 10 hours, and as the measurements stabilize (roughly 16 hours), all 4 replicates have a higher normalized fluorescence than the other strains. Consequently, we might think that Metolachlor highly activates the stress response pathway in the cells, inducing a high activation of HSP12 and thus a high production of fluorescence.

Conclusion

Here we proved that the HSP12 reporter strain that we assembled is expressing a fluorescent signal in presence of environmental stress such as Metolachlor. Furthermore, we showed that this fluorescence might be induced by the pesticides only, as the captured signal is significantly higher than the one obtained for the Methanol control, and this for the 4 replicates. As a consequence, this reporter strain could be used in our device to detect the presence of pesticides in water samples.

References

  1. Martínez-Pastor, M. T. et al. The Saccharomyces cerevisiae zinc finger proteins Msn2p and Msn4p are required for transcriptional induction through the stress response element (STRE). EMBO J 15, 2227–2235 (1996).
  2. Coleman, S. T., Epping, E. A., Steggerda, S. M. & Moye-Rowley, W. S. Yap1p Activates Gene Transcription in an Oxidant-Specific Fashion. Molecular and Cellular Biology 19, 8302–8313 (1999).
  3. Saccharomyces Genome Database | SGD [Internet]. [cited 2020 Oct 26]. Available from: https://www.yeastgenome.org/
  4. Lee, M. E., DeLoache, W. C., Cervantes, B. & Dueber, J. E. A Highly Characterized Yeast Toolkit for Modular, Multipart Assembly. ACS Synth Biol 4, 975–986 (2015).
  5. Quick and easy CRISPR engineering in Saccharomyces cerevisiae · Benchling [Internet]. [cited 2020 Oct 26]. Available from: https://benchling.com/pub/ellis-crispr-tools#parts-list
  6. Gietz, R. D. & Schiestl, R. H. Quick and easy yeast transformation using the LiAc/SS carrier DNA/PEG method. Nature Protocols 2, 35–37 (2007).

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