Team:Estonia TUIT/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).

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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 Monarch® DNA Gel Extraction Kit

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:
FavorPrep^TM Plasmid Extraction Mini Kit
Monarch^TM Plasmid Miniprep Kit

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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