iGEM 2020 — Team Estonia_TUIT

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# Team:Estonia TUIT/Notebook

Team:Estonia\_TUIT - 2020.igem.org

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# Notebook

![Responsive image](https://static.igem.org/mediawiki/2020/9/94/T--Estonia_TUIT--Notebook.jpg)

## Lab Journal Part 1

## Lab Journal Part 2

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# Team:Estonia TUIT/Engineering

Team:Estonia\_TUIT - 2020.igem.org

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# Engineering

## Overview

We aimed to create a cell factory for lipid production, where several aspects, such as the length of the cultivation cycle, the product accumulation rate and also the product extraction, were taken into account at the cell factory design stage. We started by studying the literature to find possible approaches to increase lipid production in S. cerevisiae. Many genetic modifications have been found to increase lipid accumulation, and we thought to integrate beneficial modifications from different aspects of lipid metabolism to potentially achieve a higher level of efficiency.

* [Deletions](#deletions)
* [Viability](#viability)
* [Droplets](#droplets)
* Back to top

# Evaluating the effect of TAG lipase deletions

We started by inhibiting the degradation of triacylglycerols (TAGs), as this has been shown previously to result in increased TAG accumulation (Ferreira et al., 2018). We made single, double, and triple deletion of TAG lipases *TGL3, TGL4* and *TGL5* and measured the effect of these modifications on lipid accumulation by Nile red staining of yeast. We found that the triple deletion resulted in highest lipid levels, thus we decided to continue with this strain for further modifications (Fig 1, 2).

![Responsive image](https://static.igem.org/mediawiki/2020/a/a0/T--ESTONIA_TUIT--engineering_1.png)

**Figure 1. Nile red staining shows build-up of lipids in LDs upon TAG lipase deletions.**
Microscopy images showing the cells in brightfield image and the fluorescent signal of lipids stained with Nile red. The cells are from 24 hour time point after inoculation.

![Responsive image](https://static.igem.org/mediawiki/2020/f/fb/T--ESTONIA_TUIT--engineering_2.png)

**Figure 2. TAG lipase deletions result in greatly increased lipid accumulation in LDs**. (A, B) Plots showing the mean Nile red fluorescence intensities in single cells from indicated strains. The samples were analyzed at 24 hour time point (A) and 72 hour time point (B). The bars show median and its 95% confidence intervals. \*\*\*\*, \*\*, \*, and ns note p-values <0.0001, <0.01, <0.05, and >0.05, respectively, of pair-wise comparisons with wild-type using Mann-Whitney U test.

## The effect of modifications on cell growth

In addition to increasing the stability of TAGs, we aimed to maximize the carbon flux to final product synthesis. An innovative solution for this has been suggested by Guo et al., 2018 (Guo et al., 2018), who functionalized yeast cells with indium phosphide nanoparticles to give yeast an ability to utilize solar energy for NADPH regeneration. As fatty acid synthesis is highly dependent on NADPH availability (Sheng and Feng, 2015), we hypothesized that the indium phosphide nanoparticles could be very beneficial to support the lipid production. To avoid carbon loss via NADPH regeneration, the oxidative part of the pentose phosphate pathway must be inactivated. This has been done by deletion of  *ZWF1* (Guo et al., 2018). Thus, we deleted *ZWF1* in the *tgl3Δ tgl4Δ tgl5Δ* strain.

In *S. cerevisiae,* lipids generally accumulate in the stationary phase (Werner-Washburne et al., 1993). Also, increased lipid synthesis can burden the cell metabolism and decrease cell growth. For an efficient cultivation, we wanted to separate growth and production phases. Therefore, we find it important to also check how the different genetic modifications affect the growth rate. For this, we performed growth rate assays, which revealed that although TAG lipase deletions did not have a marked effect on cell growth, the *ZWF1* deletion did (Fig 3). Based on this result, we think that our strain could be further improved by making a conditional shutoff version of *ZWF1*, instead of a conventional deletion. Interestingly, recent studies have found several efficient methods for inducible protein depletion, including light-dependent possibilities (Deng et al., 2020).

![Responsive image](https://static.igem.org/mediawiki/2020/4/42/T--ESTONIA_TUIT--engineering_3.png)

**Figure 3. Viability assay shows decreased growth rate of *zwf1Δ* strain.**

# Light-induced Plin3 promotes lipid accumulation

Not to burden the cell with lipid production during the growth phase, we wanted to create an inducible metabolic switch. We chose to use light to control this switch, as in comparison with other known inductors, light is relatively cheap, easily controllable, and does not require addition of extra compounds to the growth medium. Based on published data, we planned to overexpress three genes: *PAH1* and *DGA1*, which encode for enzymes in the TAG synthesis pathway, and human perilipin gene *PLIN3.* Plin3 has been shown to promote lipid droplet assembly, which stabilizes TAGs and enables their higher accumulation (Jacquier et al., 2013). For the inducible overexpression, we used light-regulated transcription factor VP-EL222, which is sensitive to the level on illumination and has very low background activity (Benzinger and Khammash, 2018). To mimimize the number of genetic modification steps to make the production strain, we constructed an overexpression vector that also contained the VP-EL222 transcription factor. We transformed the *tgl3Δ tgl4Δ tgl5Δ zwf1Δ* strain cells with a plasmid containing the *PLIN3* expression cassette and analyzed the lipid accumulation in these cells when grown with or without illumination (Fig 4). These experiments revealed that the lipid levels in LDs were increased upon illumination, indicating that the part we constructed has a beneficial effect on lipid production in yeast.
Also, we observed an increased lipid build-up in cells that were functionalized with the indium phosphide nanoparticles and were grown under illumination (Fig 4). This shows that the light could also be used as an energy source to drive lipid synthesis.

![Responsive image](https://static.igem.org/mediawiki/2020/c/c2/T--ESTONIA_TUIT--engineering_4.png)

**Figure 4. Light-harvesting nanoparticles and induced expression of perilipin greatly promote lipid accumulation.**  (A) Microscopy images of exemplary cells showing the accumulation of lipids in LDs detected by Nile red staining. The strains based on *tgl3Δ tgl4Δ tgl5Δ zwf1Δ* background with *PLIN3* expression plasmid or indium phosphide (InP) nanoparticles were grown with or without illumination. (B) Plot showing the average fluorescence intensities of Nile red staining. The bars show median values and its 95% confidence intervals. \*\*\*\* indicates p-value <0.0001 in pair-wise comparisons with  *PVPEL222-PLIN3* grown without illumination by Mann-Whitney U test.

# References

Benzinger, D., and Khammash, M. (2018). Pulsatile inputs achieve tunable attenuation of gene expression variability and graded multi-gene regulation. Nat. Commun. 9, 1–10.

Deng, W., Bates, J.A., Wei, H., Bartoschek, M.D., Conradt, B., and Leonhardt, H. (2020). Tunable light and drug induced depletion of target proteins. Nat. Commun. 11, 1–13.

Ferreira, R., Teixeira, P.G., Gossing, M., David, F., Siewers, V., and Nielsen, J. (2018). Metabolic engineering of Saccharomyces cerevisiae for overproduction of triacylglycerols. Metab. Eng. Commun. 6, 22–27.

Guo, J., Suástegui, M., Sakimoto, K.K., Moody, V.M., Xiao, G., Nocera, D.G., and Joshi, N.S. (2018). Light-driven fine chemical production in yeast biohybrids. Science (80-. ). 362, 813–816.

Jacquier, N., Mishra, S., Choudhary, V., and Schneiter, R. (2013). Expression of oleosin and perilipins in yeast promotes formation of lipid droplets from the endoplasmic reticulum. J. Cell Sci. 126, 5198–5209.

Sheng, J., and Feng, X. (2015). Metabolic engineering of yeast to produce fatty acid-derived biofuels: Bottlenecks and solutions. Front. Microbiol. 6, 554.

Werner-Washburne, M., Braun, E., Johnston, G.C., and Singer, R.A. (1993). Stationary Phase in the Yeast Saccharomyces cerevisiae.

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# Team:Estonia TUIT/Experiments

Team:Estonia\_TUIT - 2020.igem.org

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# Experiments

## Introduction

Protocols play a key role in uniformity of processes and creation of successful products during
experimental laboratory work. Protocols outlined here were provided to us by supervisors and sponsors
and are the basis for all experiments mentioned in the Notebook. The page includes the following
protocols: DNA polymerase chain reaction protocol (p. 1), Restriction digestion protocol (p. 2), Agarose
gel for DNA separation (p. 3), Extraction of genomic DNA from yeast cells (p. 4), DNA purification from
agarose gel (p. 5), DNA concentration measurements (p. 6), Ligation protocol (p. 6), Golden Gate
assembly (p. 7), Bacterial transformation (p. 8), Plasmid extraction from bacterial cells (p. 9-10), Plasmid
rescue from yeast (p. 11-12), Yeast transformation (p. 13-14), Yeast colony PCR (p. 15), LB and YPD
media (p. 16), Nile Red staining (p. 17), Microscopy (p. 17), Time-lapse microscopy (p. 18), Plate reader
(p. 18), InP Nanoparticles preparation (p. 19), Activation of nanoparticles (p. 20), Cell surface
modification (p. 21), Microbial biohybrid assembly (p. 22), CRISPR/Cas9 protocol (p. 23-25), Yeast
viability assay (p. 25).

1. 1
2. 2
3. 3
4. 4
5. 5
6. 6
7. 7
8. 8
9. 9
10. 10
11. 11
12. 12
13. 13

1. 14
2. 15
3. 16
4. 17
5. 18
6. 19
7. 20
8. 21
9. 22
10. 23
11. 24
12. 25

### DNA polymerase chain reaction protocol

To amplify DNA fragments for subsequent cloning, PCR with Phusion DNA polymerase was used. Components of PCR reaction are listed in **Table 1**.

All components of the PCR reaction were mixed on ice. PCR tubes with the reaction mix were transferred into a 98 °C preheated thermocycler and PCRs were performed according to the conditions listed in the **Table 2**.

Table 1 PCR reaction components and Table 2 PCR program.

| Components | 50 µL reaction | Final concentration |
| --- | --- | --- |
| Buffer 5X (HF or GC) | 10 µL | 1X |
| dNTPs mix (25 mM each) | 0.4 µL | 200 µM each |
| Forward primer (10 µM) | 2.5 µL | 0.5 µM |
| Reverse primer (10 µM) | 2.5 µL | 0.5 µM |
| DNA template: Genomic DNA,Plasmid DNA | X µL; X µL | 50 ng - 250 ng; 1 pg–10 ng |
| Phusion DNA Polymerase | 0.5 μL | 0.02 U/µL |
| Nuclease-free water | to 50 µL | - |

| Step | T(°C) | Time (s) |
| --- | --- | --- |
| Initial Denaturation | 98 | 30 |
| 25-35 Cycles | 45-72 | 20 |
| 72 | 15-30 s per kb |
| Final Extension | 72 | 5 min |
| Hold | 15 | ∞ |

### Restriction digestion protocol

All restriction enzymes used in our work were FastDigest enzymes from Thermo Fisher Scientific. Restriction reactions were prepared by mixing the following reagents **(Table 3):**

| Components | Volumes |
| --- | --- |
| Water | to 20 μL |
| 10X FastDigest Buffer | 2 μL |
| DNA | 1 μg |
| Restriction enzymes | 1 μL |
| FastAP (for vector DNA only)\* | 0.5 μL |

\*FastAP (Thermosensitive Alkaline Phosphatase) was added to the vector restriction reaction in order to prevent self-ligation of the vector without the insert.

All components of restriction reactions were mixed in a 1.5 mL tube. After mixing, the restriction reaction was incubated at 37 °C for 30-60 min. Following the incubation, restriction enzymes were inactivated by heating the mixture for 15 min at 65-85 °C. As the next step, PCR clean-up protocol or gel electrophoresis followed by DNA purification from the gel was performed to clean the restricted DNA fragments.

### Agarose gel for DNA separation

All restriction enzymes used in our work were FastDigest enzymes from Thermo Fisher Scientific. Restriction reactions were prepared by mixing the following reagents **(Table 3):**

| Ingredients |
| --- |
| TAE (trishydroxymethylaminomethane – ethanoic acid – ethylenediaminetetraacetic acid) |
| Atlas ClearSight DNA Stain |

1. In order to make 1% agarose gel, 1 g of agarose was added to 100 mL of TAE buffer solution.
2. The mixture was heated in a microwave until agarose was completely dissolved.
3. The solution was left to cool down to approximately 60 °C.
4. 6 μL of Atlas ClearSight DNA Stain was added.
5. The solution was poured into a gel caster with an appropriate comb installed.
6. After polymerization, the gel was used for DNA electrophoresis.

### Extraction of genomic DNA from yeast cells.

| Ingredients | Volumes |
| --- | --- |
| liquid yeast culture | 100-200 μL |
| lithium acetate (LiOAc), 1% SDS solution | 100 μL of 200 mM |
| 96-100% ethanol | 300 μL |
| 70% ethanol | 1 mL |
| H2O or TE buffer | 100 mL |
| Optional: | 10 μL RNAse A into 50 μL of cells |

1. Suspend cells in LiOAc-SDS solution
2. Incubate 5 min 70 °C
3. Add 96% ethanol
4. Centrifuge 15000xg 3 min, discard supernatant
5. Add 70% ethanol
6. Centrifuge 15000xg 3 min, discard supernatant
7. Dissolve the pellet in TE buffer
8. Centrifuge 15000xg 15 sec, use supernatant as template in PCR
9. Optional: Incubate with RNAse at 37 °C 1 hour

### DNA purification from agarose gel

Ingredients:

1. Minicolumns
2. R7SI agarose melting solution
3. A1 wash solution
4. Sodium Acetate (3 M, pH 5.5)
5. Isopropanol
6. TE buffer

DNA was purified from the gel using the Gel-Out kit from A&A Biotechnology according to the manual instructions. A brief description of the procedure is written below.
1. DNA fragments of the correct sizes were cut out from the gel and transferred to Eppendorf tubes.
2. R7SI agarose melting solution was added; in case of < 2% agarose gel - 400 μL, in case of ≥ 2% agarose gel - 500 μL. Samples were incubated at 50°C until the complete dissolution of agarose slices. During incubation, the
samples were mixed from time to time by inverting the tubes or vortexing.
3. An appropriate volume of Isopropanol was added.
4. < 2% agarose gel - 200 μL.
5. ≥ 2% agarose gel - 250 μL.
6. The samples were mixed by inverting the tubes.
7. After brief centrifugation, the mixture was transferred onto the minicolumns, which were centrifuged for 30 s at 10 000-15 000 RPM.
8. The flow-though was discarded and the minicolumns were re-assembled with the collection tubes.
9. At the next step, 600 μL of A1 wash solution were added and centrifugation for 30 s at 10 000-15 000 RPM was performed.
10. The filtrates were discarded and minicolumns were re-assembled with the collection tubes.
11. 300 μL of A1 wash solution were added.
12. Centrifugation for 1 min at 10 000-15 000 RPM was performed.
13. The dry minicolumns were transferred to the new 1.5 mL elution tubes and 50 μL of TE buffer were added directly onto the minicolumns resin.
14. Minicolumns were incubated for 3 min at room temperature and centrifuged for 60 s at 10 000-15 000 RPM.
15. The minicolumns were discarded. Purified DNA was used right away or kept at -20°C until needed. Additional protocols used:

[FavorPrep™ GEL/PCR Purification Kit](http://www.favorgen.com/port_pro1.php?type_1=pro_v_p11&id_1=dis15b9a6fd7ed641)
[Monarch® DNA Gel Extraction Kit](https://international.neb.com/protocols/2015/11/23/monarch-dna-gel-extraction-kit-protocol-t1020)

### DNA concentration measurements

DNA concentration measurements were carried out with NanoDrop Microvolume Spectrophotometer according to the manufacturer manuals. Software used: NanoDrop 1000 v.3.8.1.
Mode used: Nucleic Acid, DNA-50

### Ligation protocol

All ligation reactions were performed using T4 DNA ligase from NEB according to the manufacturer's instructions. All the components were mixed in a 1.5 mL tube in accordance with Table 4. DNA was taken using the 1:3 ratio of vector : insert based on the size of DNA fragments.

Table 4. Components of the ligation reaction.

| Component | Amount |
| --- | --- |
| Water | add to 20 μL |
| 10X T4 DNA Ligase Buffer | 2 μL |
| Vector | 0.020 pmol |
| Insert | 0.060 pmol |
| T4 DNA Ligase | 1 unit (5 units for blunt-end ligation) |
| PEG 4000 (for blunt-end ligation) | 2 μL |

Ligation reactions were kept at 16 °C for 2-4 hours or overnight (if ligation contains blunt ends). 1-2 µL of ligation mixture was used for bacterial transformation.

### Golden Gate assembly

Golden Gate protocol was performed using the MoClo yeast kit developed by Lee et al. (2015). The kit includes a set of characterized parts which, together with custom-made parts, allow for assembly of synthetic constructs. The Golden Gate assembly reaction was mixed together based on Table 5 and the reaction program is shown in Table 6.

Table 5. Components of Golden Gate assembly reaction

| Reagents | Assembly reaction |
| --- | --- |
| Plasmid or PCR product | 0.5 μL |
| T4 DNA Ligase buffer (NEB) | 1 μL |
| Hi-T4 DNA Ligase (NEB) | 0.5 μL |
| Type IIS restriction enzyme | 0.5 μL |

Table 6. Golden Gate reaction Thermocycler program

| step | Temp | Time |
| --- | --- | --- |
| 25 Cycles of digestion | 42 °C | 2 min |
| 16 °C | 5 min |
| Hi-T4 DNA Ligase (NEB) | 60 °C | 10 min |
| Type IIS restriction enzyme | 80 °C | 10 min |

### Bacterial transformation

Necessary components:
Escherichia coli (E. Coli) competent cells
Lysogeny Broth (LB) media

DNA transformation to bacteria cells was performed as follows:

1. Competent Escherichia coli cells were transferred from -80 °C freezer to ice. E. coli cells were left on ice for approximately 10 min to thaw.
2. 50 μL of cells was mixed with 2 μL of ligation reaction (50 pg - 1 ng for plasmid DNA).
3. Cells were kept on ice for 30 min.
4. Heat-shock was performed at 42 °C for 30 s.
5. Cells were cooled for 1-2 min on ice.
6. 1 mL of LB medium was added to the cells.
7. Tubes were incubated in the shaker at 37 °C and 220 rpm for 40 min.
8. Meanwhile, bacterial selection plates (LB with antibiotics) were transferred from the cold room to room temperature (Plates were labeled accordingly and 10-20 glass beads were added to each of them).
9. After incubation, 1.5 mL tubes with bacteria were centrifuged for 1 min at 5000 rpm.
10. Excess of supernatant was discarded (approximately 250 µL was left in the tube).
11. Bacterial cells were resuspended in the supernatant leftover and transferred on the selection plate.
12. Bacterial cells were spread on the plate surface evenly using sterile glass beads.
13. Glass beads were removed, and plates were incubated at 37 °C for 12-14 hrs (until single colonies appeared).

### Plasmid extraction from bacterial cells

*Ingredients:*

* Minicolumns
* L1 colour cell suspension solution
* L2 lysis solution
* GL3 neutralizing solution
* W first wash solution
* A1 second wash solution
* TE buffer

Plasmids were extracted from bacterial cells using the “Plasmid Mini” kit by A&A Biotechnology according to manual instructions. A brief description of the procedure is written below.

1. Up to 3 mL (1.5-3 mL) of overnight bacterial culture was centrifuged. After that supernatant was removed and the bacterial pellets were suspended in 200 µL of L1 cell suspension solution.
2. 200 µL of L2 lysis solution was added and the solution was carefully mixed by turning the 1.5 mL tube up-side-down. The mix was incubated for 3 min at room temperature.
3. 400 µL of GL3 neutralizing solution was added and carefully mixed until the disappearance of the raspberry color of the lysates.
4. The lysates were centrifuged for 10 min at 10 000-15 000 RPM.
5. The supernatants were transferred onto the minicolumns, which were centrifuged for 1 min at 10 000-15 000 RPM.
6. The minicolumns were removed from the collection tubes and the filtrates were discarded then the minicolumns were re-assembled with the same collection tubes.

7. 500 µL of W first wash solution were added and the minicolumns were centrifuged for 1 min at 10 000-15 000 RPM.
8. The minicolumns were removed from the collection tubes, the filtrates were discarded and the minicolumns re-assembled with the same collection tubes.
9. 600 µL of A1 second wash solution were added and centrifugation for 2 min at 10 000-15 000 RPM was performed.
10. The dry minicolumns were carefully transferred into new 1.5 mL tubes. 60 µL of TE buffer was added directly onto the minicolumns resin to elute DNA.
11. Minicolumns were incubated for 3 min at room temperature followed by 1 min centrifugation at 12 000 RPM.
12. Eluted plasmid DNA was used straight away or stored at -20°C until needed.

**Additional protocols used:**
[FavorPrepTM Plasmid Extraction Mini Kit](http://www.favorgen.com/favorgen/serv_1/mem_t1/h_1/pdf/plasmid/FAPDE%20004%20100%20300.pdf)
[MonarchTM Plasmid Miniprep Kit](https://international.neb.com/-/media/nebus/files/manuals/manualt1010.pdf?rev=5bc17659c6df4f9cbb585e6d6973180d&hash=926E0752DD30125061D3419C486176A6)

### Plasmid rescue from yeast

*Ingredients:*

* STET buffer
* Ammonium acetate
* 96% ethanol
* 70% ethanol
* MQ water

*Plasmid extraction from yeast was performed following protocol written bellow:*

1. Grow a 1.5 ml culture overnight under selective conditions.
2. Harvest cells by centrifugation (6000 rpm, 1 minute).
3. Resuspend the pellet in 100 µl of STET buffer and add 0.2 g of 0.45 mm glass beads.
4. Vortex the tubes vigorously for 5 minutes or use bead-beater (4.0 m/s, 40 seconds).
5. Add another 10 µl of STET and vortex the tubes briefly.
6. Incubate tube at 100 °C for 3 minutes.
7. Cool briefly on ice and spin in a centrifuge for 10 minutes at 4 °C at max RPM.
8. Transfer 100 µl of the supernatant to a new tube containing 50 µl of 7.5 M ammonium acetate.
9. Incubate at -20 °C for 1 hour.
10. Centrifuge for 10 minutes at 4 °C max RPM.
11. Add 100 µl of supernatant to 200 µl of ice-cold 96% ethanol and recover the DNA by centrifugation at max RPM.
12. Wash with 1000 µL of 70% ethanol and resuspend pellet in 20 µl of MQ water.

13. Use 10 µl of this solution to transform competent bacteria.

*STET buffer mentioned in this protocol was prepared according to such proportions:*

* 8% sucrose;
* 50 mM Tris-HCl, pH 8.0;
* 50 mM EDTA;
* 70% ethanol
* 5% Triton X-100.

### Yeast transformation

*Ingredients:*

* YPD (yeast extract peptone dextrose) medium
* 100 mM LiAc (Lithium Acetate)
* TE buffer (tris(hydroxymethyl)aminomethane - Ethylenediaminetetraacetic acid buffer)
* Salmon Sperm DNA (SS-DNA)
* PEG (Poly(ethylene glycol))/LiAc (40% PEG 3350 + 100mM LiAc in 1 x TE)
* DMSO

In order to genetically transform yeast cells, the desired yeast strain was pre-grown in 50 mL of liquid YPD until OD600 0.6-0.8. The transformation was performed based on the following protocol:

1. Cell culture was centrifuged in a sterile 50 mL falcon tube. The supernatant was discarded.
2. The cell pellet was resuspended in 1 mL of sterile 100 mM LiAc in TE and transferred into 1.5 mL tube.
3. Cells were centrifuged at 3600 rpm for 60 s. The supernatant was removed and cells were resuspended in two times of cell volume of 100 mM LiAc in TE.
4. Cells were incubated for 10 min at room temperature.
5. Single-stranded carrier DNA was prepared: Salmon Sperm DNA(SS-DNA) was boiled for 10 min at 100 °C and immediately chilled on ice before use. This ensured that the DNA was single-stranded. SS-DNA was kept on ice for the rest of the time before use.
6. 20 μL of plasmid DNA (yeast integration vector restricted with proper restriction enzyme) and 10 μL of SS-DNA was mixed in a separate 1.5 mL tube.

7. 100 μL of yeast competent cells were added to the DNA and mixed.
8. At the next step, 700 ul of sterile PEG/LiAc (40% PEG 3350 + 100mM LiAc in 1 x TE) were added.
9. Finally, 48 μL of DMSO ((CH3)2SO) was added and everything was gently mixed by pipetting.
10. The mixture was incubated at 42 °C for 40 min (heat shock).
11. After the heat shock, the cells were chilled on ice for 2 min.
12. At the next step, the cells were centrifuged for 30 s at 6000 rpm at room temperature. The supernatant was removed and the cells were resuspended in 1 mL of sterile 1x TE buffer.
13. After that, the cells were centrifuged for 60 s at 3600 rpm at room temperature. The supernatant was discarded and the cells were resuspended in 200 μL of sterile 1x TE buffer.
14. Finally, 200 μL of suspension were transferred on the yeast selection plates and cells were evenly spread all over the plate surface using sterile glass beads.
15. The plates were incubated upside down at 30 °C until colonies appeared (usually, for 2-3 days).

### Yeast colony PCR

Preparation of yeast cell lysates:

1. Cells of a single yeast colony (taken with a pipette tip) were transferred into 30 µL of 20 mM NaOH in a 1.5 mL tube.
2. The mixture was boiled at 100 °C for 10 min.
3. Lysates were placed on ice for cooling followed by 1 min centrifugation at 12000 rpm.
4. Yeast cell lysates were placed on ice.
5. 1 µL of supernatant was used as a template for PCR reaction.

Preparation of PCR mixture

Components of the PCR reaction were mixed according to **Table 7.**

| Step | Temp | Time |
| --- | --- | --- |
| Initial Denaturation | 95 °C | 5 min |
|  | 95 °C (Denaturation) 30 s |
| 25-35 Cycles | 48-60 °C (Primer annealing) | 20-35 s |
|  | 72 °C (Elongation) | 1 min/kb |
| Final Extension | 72 °C | 5 min |
| Hold | 15 °C | inf |

**Table 7. PCR program.**

| Components | Volumes (°C) |
| --- | --- |
| Water | 5 µL |
| 2X "PCR Mix Plus Green" | 12.5 µL |
| DNA template | 1 µL |
| Forward & reverse primers | 0.75 + 0.75 μL |

**Table 8.Components for PCR with “PCR Mix Plus Green” (Provided by A&A Biotechnology).**

Conditions used for yeast colony PCR are described in **Table 8.**

### LB and YPD media

Tables 9 and 10, respectively, provide list of components for preparation of LB and YPD media.

**Table 9. YPD components.**

| Components | Mass and Volumes |
| --- | --- |
| Tryptone | 10 g |
| Yeast extract | 5 g |
| NaCl | 10 g |
| H2O | up to 1L |

**Table 10. LB medium components.**

| Components | Mass and Volumes |
| --- | --- |
| Peptone | 20 g/L |
| D-Glucose anhydrous | 20 g/L |
| Micro granulated yeast extract | 10 g/L |
| Bacto agar (for plates) | 15 g/L |

1. All components were mixed in 2 L Erlenmeyer flask with a magnetic stirrer in it.
2. 15 g of Bacto Agar (for plates only, Bacto Agar was omitted for liquid medium) was added.
3. The medium was autoclaved for 15 min at 121 °C.
4. The medium was left to cool down to approximately 55 °C.
5. If necessary, 1 mL of sterile 100 mg/mL antibiotic solution was added to the medium.
6. Approx. 30 mL of medium was poured into the sterile Petri dishes and was left for cooling down and polymerization.
7. Plates were kept at +4°C for up to two weeks.

### Nile red staining

Nile red is a lipophilic stain which can be used as a lipid droplet dye and can be visualized under a fluorescence microscope. Nile red staining was performed as described by Greenspan et al. (1985).

*Ingredients:*
1 mg/mL Nile red in DMSO
Phosphate-buffered saline (PBS)

1. Centrifuge 100 µL of cell culture at 2000 x g for 1 minute
2. Remove the supernatant and resuspend the pellet in 500 µL PBS.
3. Add 5 µL 1 mg/mL Nile red solution in DMSO to the cell suspension.
4. Centrifuge the cells at 2000 x g and wash twice with 1 mL PBS.
5. Resuspend in 100 µL PBS.

### Microscopy

For the promoter characterization part, yeast colonies were checked after the transformation for the presence of GFP fluorescence. Prior to the experiment, yeast cultures were grown in synthetic complete uracil dropout medium with 2% glucose as a carbon source. The optical density (OD600) was in the range 0.2-0.6. Cultures were imaged under a Zeiss Axio Observer Z1 microscope. The following settings were used for the imaging: 25 ms exposure time for brightfield or phase-contrast, 100 ms exposure time for EGFP channel. Colibri LED modules were used as a source of 470 nm excitation at 25% intensity. Nile red was excited with the 470 nm LED for 5 ms. The fluorescence intensities were quantified using ImageJ.

### Time-lapse microscopy

Prior to time-lapse microscopy, the yeast cells were grown in synthetic complete (SC) media lacking histidine or SC lacking histidine and tryptophan depending on the integrative plasmids in the strain. The cultures were grown to OD600 0.2-0.8, the cells were pipetted onto a 0.08 mm cover glass slip and were covered with 1.5% agar-SC. Zeiss Observer Z1 microscope with 63C/1.4NA oil immersion objective and Axiocam 506 mono camera was used for imaging. The focus was kept using Definite Focus and the sample was kept at 30 °C using PeCon TempControl 37-2 digital. The cells were imaged every 5 minutes using brightfield illumination and the experiments were 13-18 hours long.

### Plate reader

Plate readers are instruments that are used to detect samples in microtiter plates. We used BioTek Synergy MX microplate reader to measure the cells’ optical density and intensity of EGFP fluorescence. All strains used in the experiments were pre-grown overnight at 30 °C in liquid CSM/2%Glc media and diluted to OD600 in a range of 0.9 to 1.1. Then diluted cell cultures were distributed to 96 well plates (clear flat bottom). For replicates of two colonies from each strain were analyzed. 200 µL of cells were added to each well. CSM/2%Glc media was used as a reference to OD600. The measurements were done with the following parameters. Absorbance was measured at 600 nm and fluorescence was measured at the following conditions: excitation 485 nm, emission 528 nm, bandwidth 20, gain 80.

### InP Nanoparticles preparation

*Ingredients:*
MQ water
InP piece

1. Hammer the InP into small pieces.
2. Grind with mortar and pestle in water solution until you do not feel any particles with the mortar (ca. 2h for around 2.5 g piece).
3. Transfer this nanoparticle water suspension into two 5 ml tubes.
4. Ultrasonicate to separate particles that have aggregated.
5. Dilute in 50 ml of distilled water.
6. Centrifuge at 8000x g for 5 min.
7. Big particles remain on the walls of the flask, the suspension is transferred into a new flask.
8. Centrifuge at 8000x g for 5 min.
9. Resuspend the remaining particles from step 7 in 50 ml distilled water. Sonicate and centrifuge at 8000x g for 5 min. Collect both supernatants from steps 8 and 9 into a new tube.
10. Centrifuge at 10000x g for 15 min.
11. Transfer suspension into a new bigger tube and add additional 50 ml of water.
12. Centrifuge at 8000x g for 5 min.
13. Transfer suspension into a new tube.
14. Centrifuge at 10000x g for 15 min.
15. Transfer suspension into a new tube.
16. Centrifuge at 8000x g for 5 min.
17. Transfer suspension into a new tube.
18. Centrifuge at 10000x g for 15 min
19. The nanoparticles are now on the tube wall, and are collected by wall scraping, and further transferred into 5 ml tube and resuspended in distilled water.

### Activation of nanoparticles

*Ingredients:*
MQ water
FeCl3∙6H2O (5 mg mL-1)
Tannic acid (40 mg mL-1)
Tris-HCl buffer (pH 8.0, 1M)
70% ethanol

1. Suspend nanoparticles in MQ water to 0.5 – 5% w/v.
2. Transfer 500 µL of suspension into a new 2 ml tube.
3. Add 500 µL of FeCl3∙6H2O (5 mg mL-1) and 500 µL of tannic acid (40 mg mL-1). Mix by pipetting up and down.
4. Add 60 µL of Tris buffer (pH 8.0, 1M). Slowly add it till pH of the solution becomes 8!
5. Spin down nanoparticles at 8,000 g, 5 min.
6. Remove supernatant. Wash the pellet 3-4 times with MQ water at 8,000 g, 5 min.
7. After the wash, add 1 mL of 70% ethanol and incubate at room temperature for 10 min.
8. Centrifuge at 8,000 g, 5 min. Repeat step 6 in full.
9. After final wash, resuspend nanoparticles in 1 mL MQ water.
10. Sonicate the suspension before use.

### Cell surface modification

*Ingredients:*

MQ water
Polyallylamine hydrochloride (PAH) (5 mg mL-1)

1. Wash the grown cell culture in MQ water 3-4 times. Centrifugation is performed at 3,000g, 2 min.
2. Resuspend cells in 500 µL of MQ water to achieve OD600 1.6 ~ 3.2.
3. Dilute and transfer 450 µL of cell suspension. Recommended OD600 1.6 ~ 3.2.
4. Add 50 µL of PAH (polyallylamine hydrochloride) and gently vortex for 20 ~ 40 seconds before spinning down and removing the supernatant.
5. Wash the pellet in MQ water 3-4 times. Centrifugation is performed at 3,000g, 2 min.
6. Resuspend yeast in MQ water to achieve desirable OD600.

### Microbial biohybrid assembly

*Ingredients:*

FeCl3∙6H2O (5 mg mL-1)
PBS (pH 7.4)
MQ water

1. Use OD600 cell to nanoparticles ratio 1.6 ~ 2.0 for the assembly.
2. Mix cells with nanoparticles in a new 1.5 mL tube. Vortex mix for 10-60 s.
3. Add FeCl3∙6H2O (5 mg mL-1) in a relation of 0.01 : 1 to full volume of mix in step 2.
4. Add an equal amount of PBS (pH 7.4) as volume of mix from step 2.
5. Wash final mix with MQ water 3 times at 2,000 g, 2 min.
6. Transfer mix into selected growth medium.

### CRISPR/Cas9 protocol

We used the Ellis Lab CRISPR/Cas9 toolkit as described by William Shaw. This method allows for markerless integration of multiple genetic cassettes into selected genomic loci. The toolkit consists of:

An sgRNA entry vector
A range of Cas9-sgRNA expression vectors
Markerless integration cassettes

gRNAs targeting ACC1 region were designed using the Benchling CRISPR Guide RNA design tool. Four gRNAs with the highest on- and lowest off-target scores were chosen, and 5’ overhangs were added for cloning into a BsmBI-linearized plasmid. The following step describes the process of gRNA assembly and cloning.

Phosphorylation of oligos (inserts need to have 5’ phosphates for ligation, but synthesized primers do not have this).

| Components | Volumes |
| --- | --- |
| 10x T4 DNA ligase buffer (Thermo Scientific) | 1 µL |
| sgRNA oligo 100 µM | 1 µL |
| T4 PNK | 1 µL |
| H2O | 7 µL |

The mixture was incubated 1h at 37 °C for phosphorylation reaction. Then PNK was inactivated at 75 °C for 10 min.

1. For annealing of oligos, 10 µL of each of the two of phosphorylated oligos were mixed and 180 µL of MQ H2O was added. 50 µL of this mix was run in the thermocycler with the following program: 96 °C for 6 min, followed by a decrease in increments of 0.1 °C per second, down to 23 °C.
2. For ligation of the annealed oligos to sgRNA entry vector, the following mix was prepared and kept at room temperature for 15 min. The resulting mix was

| Components | Volumes |
| --- | --- |
| Annealed oligos | 0.5 µL |
| BsmBI-linearized pWS082 | 0.5 µL |
| 10x T4 ligase buffer (Thermo Scientific) | 1 µL |
| T4 DNA Ligase | 0.5 µL |
| H2O | 7.5 µL |

| Components | Volumes |
| --- | --- |
| sgRNA vector | 1 µg |
| 10x FastDigest buffer | 1 µL |
| Eco32I | 0.5 µL |
| H2O | Up to 10 µL |

Once assembled and sequence verified, the sgRNA expression cassette was linearized with FastDigest Eco32I.

The mix was incubated at 37 °C for 30 min.

For genome editing, the following mix was prepared and used in the Super-High Efficiency yeast transformation protocol provided by Ellis Lab.

| Components | Transformation reaction |
| --- | --- |
| Linearized Cas9-gRNA gaprepair vector | 100 ng |
| Each digested sgRNA vector | 200 ng |
| PCR amplified donor DNA | > 2 µg |

For transformation, we used 2 µL of the linearized Cas9-sgRNA repair vector (pWS171), 2 µL of linearized sgRNA cassette, and 36 µL of ACC1S686A S659A S1157A purified PCR product

### Yeast viability assay

The cultures were diluted to 106 cells/mL. Then, this culture was diluted 2, 10, 100 and 500 times. 5 µL of the dilution series was plated on YPD plates. The plated were incubated overnight at 30 °C.

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# Team:Estonia TUIT/Results

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# Results

## Overview

With the aim to create a lipid-producing cell factory, we designed a set of strains with modifications to improve various aspects of lipid metabolism. We show that decreasing TAG turnover by deletion of TAG lipases results in increased lipid levels. Light-activated expression of perilipin promotes lipid build-up, presumably by sequestering TAG to lipid droplets. Finally, we show that indium phosphide nanoparticles can functionalize yeast cells to use light energy for lipid synthesis.

* [Lipase](#lipase)
* [Growth rate](#growth_rate)
* [Perilipin](#perilioin)
* [Biohybrids](#biohybrids)
* [Future plans](#part4)
* [References](#part5)
* Back to top

## Lipase deletions lead to increased lipid accumulation

First, we studied the effects of different TAG lipase (*TGL3, TGL4, TGL5*) and *ZWF1* gene deletions on lipid production in a batch culture experiment. The strains were inoculated in a liquid medium at equal densities and the lipid accumulation was monitored by Nile Red staining after 24 and 72 hours of cultivation. Nile red is a fluorescent dye that interacts specifically with neutral lipids in lipid droplets (LDs) and it does not stain the cellular membranes (Greenspan et al., 1985). In 24 hours after dilution, the cells from wild-type strain showed very low Nile red staining, indicative of low intracellular lipid levels (Fig 1, 2A). Single deletions of *TGL3* and *TGL4* resulted in a considerable increase in lipid accumulation, with *tgl3Δ* having a slightly greater effect than *tgl4Δ* (Fig 1, 2A). Interestingly, in the cells with TAG lipase deletions, clearly visible LDs could be detected in both the bright-field image and the Nile Red fluorescent image (Fig 1). Although *tgl3Δ tgl4Δ* double deletion did not further increase the lipid levels in LDs, a triple deletion of all major TAG lipases (*tgl3Δ tgl4Δ tgl5Δ*) had a profound positive effect on lipid build-up. In the 24-hour time point, the Nile Red staining intensity of the triple TAG lipase deletion strain was not affected by the deletion of *ZWF1* (Fig 1, 2A).

The lipid accumulation was also measured at 72 hours following dilution. In this time point, the culture had reached the stationary phase, and LDs were clearly detectable with Nile red staining for all tested strains, including the wild-type (Fig 2B). Lipid accumulation is known to increase in  *S. cerevisiae cells*  upon nutrient depletion (Werner-Washburne et al., 1993), and our results reveal that the difference between wild-type and TAG lipase deletion strains is considerably reduced in the stationary phase cells (Fig 2B). In the 72-hour time point, we did not observe a statistically significant difference between wild-type and the single  *tgl4Δ*  or double  *tgl3Δ tgl4Δ*  deletion cells. Single deletion of *TGL3*, which had the largest effect in the 24-hour time point, also led to increased LD staining intensity at 72 hours. We observed the highest intracellular lipid levels with the triple  *tgl3Δ tgl4Δ tgl5Δ*  deletion strain, and interestingly, in this time point, the  *zwf1Δ*  strain had considerably lower LD staining, comparable to the level of the wild-type strain (Fig 2B). Although the lipid levels of  *zwf1Δ*  strain were not lower in the 24-hour time point,  *ZWF1*  deletion could be expected to result in decreased lipid synthesis, as Zwf1 is required to regenerate NADPH, a critical cofactor in fatty acid synthesis. Taken together, by preventing TAG degradation with triple deletion of TAG lipases, we have achieved a considerable increase in lipid production compared to the wild-type strain.

![Responsive image](https://static.igem.org/mediawiki/2020/8/81/T--ESTONIA_TUIT--engin_fig1.jpg)

Figure 1. Nile red staining shows build-up of lipids in LDs upon TAG lipase deletions. Microscopy images showing the cells in brightfield image and the fluorescent signal of lipids stained with Nile red. The cells are from 24 hour time point after inoculation.

![Responsive image](https://static.igem.org/mediawiki/2020/archive/7/76/20201025233458%21T--EStonia_TUIT--engenering_fig2.png)

Figure 2. TAG lipase deletions result in greatly increased lipid accumulation in LDs. (A, B) Plots showing the mean Nile red fluorescence intensities in single cells from indicated strains. The samples were analyzed at 24 hour time point (A) and 72 hour time point (B). The bars show median and its 95% confidence intervals. \*\*\*\*, \*\*, \*, and ns note p-values <0.0001, <0.01, <0.05, and >0.05, respectively, of pair-wise comparisons with wild-type using Mann-Whitney U test.

# *ZWF1* deletion decreases growth rate

We aimed to optimize the production process so that the growth phase (biomass accumulation) would not be negatively affected by simultaneous product synthesis. This would allow rapid biomass growth and shorter production cycles. Thus, we were interested to test whether the modifications in our production strain affect the growth rate. For this, we tested the effect of TAG lipase and *ZWF1* deletions in a serial dilution viability assay. The experiment showed that while TAG lipase deletions did not significantly affect the growth rate, the *ZWF1* deletion did reduce the growth rate (Fig 3). This suggests that the production process could be made more efficient by triggering inducible shutoff of Zwf1 at a time when sufficient biomass has been reached. In this case, Zwf1 would be present during the growth phase, allowing normal growth rate, but would be inactivated at the same time when the lipid synthesizing enzymes are induced.

![Responsive image](https://static.igem.org/mediawiki/2020/2/28/T--EStonia_TUIT--engenering_fig3.png)

Figure 3. Viability assay shows a decreased growth rate of  *zwf1Δ*  strain.

# Expression of human perilipin Plin3 results in higher intracellular lipid levels

As our next step to improve lipid bioproduction, we tested whether expression of human perilipin Plin3 could further increase the lipid bioproduction in the  *tgl3Δ tgl4Δ tgl5Δ zwf1Δ*  strain. For this, we constructed and transformed to these cells a plasmid that contains the light-inducible transcription factor VP-EL222 expression cassette and the  *PLIN3*  expression cassette with the light-regulated promoter. The cells containing this plasmid were set to grow at 30 °C either under constant illumination or in the dark. In 24 hours, the lipid accumulation in LDs was monitored by Nile red staining. Interestingly, we detected significantly higher lipid levels in the cells that were grown under illumination, which activates  *PLIN3* expression via VP-EL222 (Fig 4). As Plin3 is expected to promote LD assembly, the observed effect could be due to stabilization of the synthesized TAGs. We did not observe any noticeable changes in LD morphology between cells that either express or do not express Plin3 (Fig 4A). These results show that the lipid bioproduction in  *S. cerevisiae*  could be further improved by expression of human perilipin Plin3.

![Responsive image](https://static.igem.org/mediawiki/2020/6/68/T--EStonia_TUIT--Results_figure4.png)

Figure 4. Light-induced expression of perilipin greatly promotes lipid accumulation. (A) Microscopy images of exemplary cells showing the accumulation of lipids in LDs detected by Nile red staining.  *tgl3Δ tgl4Δ tgl5Δ zwf1Δ*  cells with  *PLIN3*  expression plasmid were grown with or without illumination. (B) Plot showing the average fluorescence intensities of Nile red staining. The bars show median values and its 95% confidence intervals. \*\*\*\* indicates p-value <0.0001 in pair-wise comparison by Mann-Whitney U test.

# Yeast biohybrids functionalized with nanoparticles use light to support lipid biosynthesis

To maximize the carbon yield in lipid production, we set out to engineer yeast biohybrids that could take use of solar power to drive their metabolism. For this, we used indium phosphide nanoparticles, which, as previously shown, when assembled on the cell, can provide electrons for NADPH regeneration (Guo et al., 2018). As NADPH is a critical cofactor for fatty acid synthesis, we hypothesized that these nanoparticles could also support lipid synthesis.
To test this, we coated the cells from  *tgl3Δ tgl4Δ tgl5Δ zwf1Δ*  strain with the indium phosphide nanoparticles and monitored their lipid accumulation when grown either in darkness or under constant illumination. At 24 hours after inoculation, we measured the lipid levels by Nile red staining. We found that the biohybrids grown under light contained substantially more intracellular lipids than the ones grown in darkness (Fig 5). This indicates that our production strain is able to harvest the light energy and direct it to lipid synthesis.
Thus, we have made significant improvements to TAG accumulation in yeast by combining three different engineering approaches. First, we showed that deletion of TAG lipases results in increased lipid levels in LDs. Secondly, we found that expression of human perilipin Plin3 in yeast leads to lipid accumulation, presumably by promoting LD assembly and stabilizing TAGs. Thirdly, we used nanoparticles to give our production strain a new quality – the ability to use light energy to drive the lipid synthesis.

![Responsive image](https://static.igem.org/mediawiki/2020/b/bc/T--EStonia_TUIT--Results_fig5.png)

Figure 5. Biohybrids use light energy to drive lipid synthesis. (A) Microscopy images of exemplary cells showing the accumulation of lipids in LDs detected by Nile red staining. The cells from  *tgl3Δ tgl4Δ tgl5Δ zwf1Δ*  background and functionalized with indium phosphide (InP) nanoparticles were grown with or without illumination. (B) Plot showing the average fluorescence intensities of Nile red staining. The bars show median values and its 95% confidence intervals. \*\*\*\* indicates p-value <0.0001 in pair-wise comparison by Mann-Whitney U test.

# Future plans

To further increase the lipid production, we aim to introduce cassettes for light-inducible
expression of  *PAH1 and DGA1* . These genes encode for enzymes that are involved in TAG
biosynthesis and it has been shown previously that their overexpression leads to increased TAG
accumulation (Ferreira et al., 2018) . Further, we plan to increase the precursor supply by
removing the feedback inhibition of Acc1, which catalyzes the formation of malonyl-CoA, a
precursor for fatty acid synthesis (Chen et al., 2018) .
In our experiments, we found that  *ZWF1*  deletion caused decreased growth rate (Fig 3).
To optimize the production process, we plan to use an inducible depletion strategy to remove
Zwf1 protein from the cells at the same time as the metabolic switch to trigger lipid synthesis is
induced. Recent studies have developed light-triggered depletion methods (Deng et al., 2020) ,
which would be an efficient solution for our strain. After introduction of every modification to
our production strain, we will measure the lipid production to test if the implemented designs
have the expected effects.

We observed a beneficial effect of functionalizing the cells with light-harvesting indium
phosphide nanoparticles (Fig 4). Although using the yeast-nanoparticle biohybrids enables
maximization of carbon yields, we find that at the moment there are several limitations to using
the indium phosphide nanoparticles in a large-scale production. First, the process of creating
the biohybrids is rather complex. Secondly, indium phosphide is an expensive compound, so it
would be necessary to recycle the nanoparticles to decrease the production costs. In fact, we
have discussed this issue with Prof. Guo, who confirmed that the nanoparticles could be
recycled. In order to increase the competitiveness of this approach, we plan to further study
different methods that enable functionalization of cells with nanoparticles, keeping in mind
that the method should be easily upscaled.

Finally, as the product extraction often makes up 85% of total production costs in
biosynthesis, we plan to engineer our production strain cells to be able to autolyse upon
induction. For this, we aim to improve our last years project, where inducible autolysis was
acheived by expression of bacterial glucanases. We hypothesize that by targeting the
glucanases directly to the cell wall, the local concentration of glucanases will reach higher
levels, necessary for the breakdown of yeast cell wall.

# References

Chen, X., Yang, X., Shen, Y., Hou, J., and Bao, X. (2018). Screening phosphorylation site
mutations in yeast acetyl-CoA carboxylase using malonyl-CoA sensor to improve malonyl-CoA-
derived product. Front. Microbiol. 9.

Deng, W., Bates, J.A., Wei, H., Bartoschek, M.D., Conradt, B., and Leonhardt, H. (2020). Tunable
light and drug induced depletion of target proteins. Nat. Commun. 11, 1–13.

Ferreira, R., Teixeira, P.G., Gossing, M., David, F., Siewers, V., and Nielsen, J. (2018). Metabolic
engineering of Saccharomyces cerevisiae for overproduction of triacylglycerols. Metab. Eng.
Commun. 6, 22–27.

Greenspan, P., Mayer, E.P., and Fowler, S.D. (1985). Nile red: A selective fluorescent stain for
intracellular lipid droplets. J. Cell Biol. 100, 965–973.

Guo, J., Suástegui, M., Sakimoto, K.K., Moody, V.M., Xiao, G., Nocera, D.G., and Joshi, N.S.
(2018). Light-driven fine chemical production in yeast biohybrids. Science (80-. ). 362, 813–816.

Werner-Washburne, M., Braun, E., Johnston, G.C., and Singer, R.A. (1993). Stationary Phase in
the Yeast Saccharomyces cerevisiae.

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