Experiments

Add 125µl 100% glycerol to 375µl liquid culture, store at -20° C

For 1 liter:

• 10g Tryptone
• 5g Yeast extract
• 10g NaCl
• 900ml ddH2O

Fill up to 1 l by adding ddH2O, autoclave.

For selective medium: Add 100 µl of 100ng/µl stock of ampicilin or 50ng/µl stock of streptomycin to 100 ml of LB

For plates: Add 2.5% w/v Agar to medium, heat in microwave until completely liquid, add antibiotic if needed, pour plates (34 ml/plate needed)

Based on original protocol by Steven Burgess 2016. Lysogeny Broth (LB). protocols.io

For 1 liter:

• 10g Yeast extract
• 10g peptone
• 900ml ddH2O
• 40mg adenine

After autoclaving, add 100 ml of sterile 20% w/v glucose

For plates: Add 2.5% w/v Agar to medium, heat in microwave until completely liquid, add antibiotic if needed, pour plates (34 ml/plate needed)

For 1 liter:

• 1.9g Yeast nitrogen base without amino acids and ammonium sulphate by FORMEDIUM
• 2g Synthetic complete mixture (KAISER), drop-out complete by FORMEDIUM
• 900ml ddH2O

After autoclaving, add 100 ml of sterile 20% w/v glucose

For 1 liter:

• 1.9g Yeast nitrogen base without amino acids and ammonium sulphate by FORMEDIUM
• 1g MSG (L-Glutamic acid monosodium salt)
• 2g Synthetic complete mixture (KAISER), drop-out complete by FORMEDIUM
• 900ml ddH2O

After autoclaving, add 100 ml of sterile 20% w/v glucose

For 1 liter:

• 1.9g Yeast nitrogen base without amino acids and ammonium sulphate by FORMEDIUM
• 1g MSG (L-Glutamic acid monosodium salt)
• 2g Synthetic complete mixture (KAISER), drop-out without uracil by FORMEDIUM
• 900ml ddH2O

After autoclaving, add 100 ml of sterile 20% w/v glucose

For plates: Add 2.5% w/v Agar to medium, heat in microwave until completely liquid, add antibiotic if needed, pour plates (34 ml/plate needed)

For 1 liter:

• 200ml M9 salts (5x stock solution)
• 1ml 1M MgSO4
• 1ml 0.1M CaCl2
• 20g Glucose
• 900ml ddH2O

30° C, shaking for:

• Saccharomyces cerevisiae BY4741
• Saccharomyces cerevisiae BY4743
• Pseudomonas putdia 1320
• Pseudomonas putdia 1243
• Pseudomonas protegens Pf-5

37° C, shaking for:

• Escherichia coli DH5-Alpha
• Escherichia coli Nissle 1917

Materials

1M CaCl2

• 3ml 1M CaCl2
• 100ml ddH2O

Filter sterilize and keep on ice.

0.1M CaCl2

Dilute 1M CaCl2 1:10 in ddH2O.

0.1M CaCl2 + 15% glycerol

• 3ml 1M CaCl2
• 4.5ml sterile glycerol
• 22.5ml ddH2O

Filter sterilize and keep on ice.

Protocol

1. Dilute O/N cultures to OD600 0.5
2. Cool culture on ice for 20 min.
3. Centrifuge at 4° C at 4000 rpm for 10 min.
4. Discard supernatant and resuspend in 20ml 0.1M CaCl2.
5. Incubate 30 min on ice.
6. Centrifuge at 4° C at 4000 rpm for 10 min.
7. Discard supernatant and resuspend all pellets in 5ml 0.1M CaCl2 + 15% glycerol.
8. Aliquot 200 µl and store at -80°C (50 µl used per transformation).

Based on original protocol by Chang et al., JEMI Methods, Vol. 1:22-25 (2017)

1. Add 1 µl of DNA to 50 µl thawed heat-shock competent cells.
2. Heat-shock for 90 sec at 42°C.
3. Add 100 µl SOC recovery medium from NEB, mix.
4. Streak on a pre-warmed plate, incubate plate at 37°C over night.

1. Add 1 µl of DNA to 50 µl thawed heat-shock competent cells.
2. Incubate for 2 min on ice.
3. Heat-shock 90 sec at 42°C.
4. Incubate for 2 min on ice.
5. Add 100 µl SOC recovery medium from NEB.
6. Incubate at 37°C shaking for 20h.
7. Streak on a pre-warmed plate, incubate plate at 37°C over night.

1. Add 2 µl of DNA to 50 µl thawed heat-shock competent cells.
2. Incubate for 30 min on ice.
3. Heat-shock 30 sec at 42°C.
4. Incubate for 2 min on ice.
5. Add 950 µl SOC recovery medium from NEB.
6. Incubate at 37°C shaking for 1h. Preheat plates to 37°C.
7. Streak 200 µl per plate, incubate plate at 37°C over night.

Materials

0.2M MOPS buffer

• 2.093g MOPS free acid
• 0.34g Sodium acetate trihydrate
• 0.146g EDTA
• 40ml ddH2O

Adjust to pH 7 using NaOH, fill up to 50 ml with ddH2O.

Filter sterilize and keep on ice.

0.2M MOPS + 15% glycerol

for 50ml:

• 250 µl 0.2M MOPS buffer
• 15ml sterile glycerol

Fill up to 50 ml with ddH2O and keep on ice.

Protocol

1. Dilute O/N cultures to OD600 0.5
2. Centrifuge 2.5 ml at 4° C at 4000 rpm for 10 min.
3. Discard supernatant and resuspend in 10 ml ice-cold ddH2O or 0.2M MOPS + 15% glycerol.
4. Repeat this wash.
5. Centrifuge 1 ml at 4° C at 4000 rpm for 10 min and resuspend in 50 µl ddH2O.
6. Use directly for electroporation.

Based on protocol by Tanaïs Moreau.

1. Add 100 ng of plasmid DNA to 50 µl electro-competent cells.
2. Transfer to a 0.1 cm cuvette.
3. Electroporate at 1.8 kV, 200 Ω, 25 µF.
4. Immediately add 200 µl LB.
5. Incubate at 37°C shaking for 2h. Preheat plates to 37°C.
6. Streak 200 µl per plate, incubate plate at 37°C over night.

Materials

TE 10x pH 7.5

• 1.21 g Tris Base
• 0.37 g EDTA

dissolve in 100 ml ddH2O and adjust pH to 7.5 with HCl

autoclave

LiAc 10x (1M) pH 7.5

• 6.99 g LiAc

dissolve in 100 ml ddH2O and adjust pH to 7.5 with HCl

autoclave

PEG4000 50%

• 2.5 g platelets of PEG4000 (PEG3350 also works)
• 1.5 ml ddH2O

mix thoroughly to dissolve and fill up to 5 ml using ddH2O

autoclave

sterile DMSO

Carrier DNA (10 mg/ml)

Salmon Sperm DNA Solution from Life Technologies/Invitrogen (Product No. 15632-011), heat denaturation by heating at 100 °C for 5 min and putting on ice immediately

Solution A (TE1x, LiAc 0.1M)

• 800 µl sterile ddH2O
• 100 µl TE10x
• 100 µl LiAc10x (1M)

Solution B (TE1x, LiAc 0.1M)

• 800 µl PEG4000 50%
• 100 µl TE10x
• 100 µl LiAc10x (1M)

Protocol

Prepare LiAc competent cells

• Per reaction, add a heaped loop full of cells (2-3 days old colony) to an Eppendorf tube containing 30 µl of solution A (TE1x, 0.1M LiAc)
• Vortex to resuspend

Transformation of LiAc competent cells

1. Add 1-3 µl plasmid DNA [50-100 ng] (the purer the DNA the less you need), mix
2. Do not forget to include a negative control without plasmid DNA and a positive control e.g. an empty vector. Latter control allows you to determine your transformation efficiency (see 'Further Information')
3. add 5 µl carrier DNA [10 mg/ml], heat denatured, mix.
4. Add 300 µl transformation solution B (TE1x, LiAc 0.1 M, PEG4000 40%),
5. Mix well (vortex).
6. Incubate for 1 h at 30°C.
7. Add 35 µl DMSO, vortex.
8. Heat shock cells for 10 min at 42 °C.
9. Cool down cells immediately on ice for 3 min
10. Spin down cells for 5 min at 800 g in an Eppendorf centrifuge.
11. Remove supernatant by pipetting.
12. Resuspend pellet very carefully in 200 µl SD-all medium.
13. Incubate cells at 30°C for 1 h.
14. Plate cells on selective medium and incubate at 30 °C (first transformants should be visible after 2 days).

Based on original protocol by Dr. Emmanuel Matabaro form our lab.

1. Pick one colony of 2-3 days old BY4741 yeast, suspend in 30 µL Zymolase.
2. Incubate at 37°C for 45 min.
3. Amplify fragment by PCR.

• 1 μl DNA (zymolased yeast)
• 1.25 μl forward primer (50 ng/μl)
• 1.25 μl reverse primer (50 ng/μl)

Add ddH2O to reach 12.5 μl, then add 12.5 μl 2x Q5® High-Fidelity DNA Polymerase mix.

Run the following PCR program:

STEP	TEMP	TIME
Initial Denaturation	98 °C	7 min
5 cycles	98 °C	10 sec
	59 °C, determined using NEB Tm calculator	30 sec
	72 °C	30 sec/kb
25 cycles	98 °C	10 sec
	72°C	30 sec
	72°C	30 sec/kb
Final Extension	72°C	2 minutes
Hold	4 °C

Based on original protocol by NEB Q5® High-Fidelity 2X Master Mix NEB

• 2 μl plasmid
• 1.25 μl forward primer (50 ng/μl)
• 1.25 μl reverse primer (50 ng/μl)

Add ddH2O to reach 12.5 μl, then add 12.5 μl 2x OptiTAQ DNA Polymerase mix.

Run the following PCR program:

STEP	TEMP	TIME
Initial Denaturation	95 °C	5 min
25-35 Cycles	95 °C	5-10 sec
	50-68 °C, determined using NEB Tm calculator	30 sec
	72 °C	1 min/kb
Final Extension	72 °C	7 minutes
Hold	4 °C

• 2 μl plasmid
• 1.25 μl forward primer (50 ng/μl)
• 1.25 μl reverse primer (50 ng/μl)

Add ddH2O to reach 12.5 μl, then add 12.5 μl 2x Q5® High-Fidelity DNA Polymerase mix.

Run the following PCR program:

STEP	TEMP	TIME
Initial Denaturation	98 °C	30 sec
25-35 Cycles	98 °C	5-10 sec
	50-72 °C, determined using NEB Tm calculator	10-30 sec
	72 °C	20-30 sec/kb
Final Extension	72 °C	2 minutes
Hold	4 °C

1. Harvest 2ml O/N bacterial culture by centrifugation at 6800 × g in a microcentrifuge for 2 min at room temperature. Decant the supernatant and remove all remaining medium.
2. Resuspend the pelleted cells in 250 μl of the Resuspension Solution. Transfer the cell suspension to a microcentrifuge tube. The bacteria should be resuspended completely by vortexing or pipetting up and down until no cell clumps remain.
3. Add 250 μl of the Lysis Solution and mix thoroughly by inverting the tube 4-6 times until the solution becomes viscous and slightly clear.
4. Add 350 μl of the Neutralization Solution and mix immediately and thoroughly by inverting the tube 4-6 times.
5. Centrifuge at 16000 × g for 5 min to pellet cell debris and chromosomal DNA.
6. Transfer the supernatant to GeneJET spin column by decanting or pipetting. Avoid disturbing or transferring the white precipitate.
7. Centrifuge for 1 min. Discard the flow-through and place the column back into the same collection tube.
8. Add 500 μl of the Wash Solution (diluted with ethanol prior to first use) to the GeneJET spin column. Centrifuge at 16000 × g for 60 seconds and discard the flow-through. Place the column back into the same collection tube. Repeat the wash procedure using 500 μl of the Wash Solution.
9. Discard the flow-through and centrifuge for an additional 1 min to remove residual Wash Solution. This step is essential to avoid residual ethanol in plasmid preps.
10. Transfer the GeneJET spin column into a fresh 1.5 mL microcentrifuge tube. Add 20 μl of the Elution Buffer to the center of GeneJET spin column membrane to elute the plasmid DNA. Take care not to contact the membrane with the pipette tip. Incubate for 2 min at room temperature and centrifuge for 2 min.
11. Measure concentration using Nanodrop and store at -20° C.

1. Dissolve 1-2% w/v Agarose in TAE buffer by heating in a microwave.
2. Add 0.1% v/v SYBR Safe to the solution & mix.
3. Pour gel into prepared tray.
4. Remove comb inside TAE buffer.
5. Mix PCR sample with 6x loading dye without SDS.
6. Load 5 μl GeneRuler ladder (10 kb) and samples in separate wells.
7. Run electrophoresis at 140 V and 400 mA for 20 min.
8. Inspect gel under UV light.

Gel purification:

1. Following electrophoresis, excise DNA band from gel precisely under UV light and transfer to a 1.5 ml microcentrifuge tube.
2. Add 10 μl of Membrane Binding Solution per 10 mg of gel slice. Vortex and incubate at 50-65 °C until gel slice is completely dissolved.

PCR cleanup:

1. Add an equal volume of Membrane Binding Solution to the PCR amplification.

1. Insert SV Minicolumn into Collection Tube.
2. Transfer dissolved gel mixture or prepared PCR product to the Minicolumn assembly. Incubate at room temperature for 1 minute.
3. Centrifuge at 16,000 × g for 1 minute. Discard flowthrough and reinsert Minicolumn into Collection Tube.
4. Add 700μl of Membrane Wash Solution (ethanol added). Centrifuge at 16,000 × g for 1 minute. Discard flowthrough and reinsert Minicolumn into Collection Tube.
5. Repeat wash with 500μl of Membrane Wash Solution. Centrifuge at 16,000 × g for 5 minutes.
6. Empty the Collection Tube and recentrifuge the column assembly for 1 minute with the microcentrifuge lid open to allow evaporation of any residual ethanol.
7. Carefully transfer Minicolumn to a new 1.5 ml microcentrifuge tube.
8. Add 50μl of Nuclease-Free Water to the Minicolumn. Incubate at room temperature for 1 minute. Centrifuge at 16,000 × g for 1 minute.
9. Measure concentration using Nanodrop and store at -20° C.

• up to 17 μl plasmid
• 2 μl 10x NEB rCutSmart™ Buffer
• 1 μl of each restriction enzyme

Add ddH2O to reach 20 μl, incubate at 37°C for 60 min.

Gel purify or heat-inactivate restriction enzymes.

Work on ice. Higher manganese concentrations introduce more mutations but decrease PCR yield.

• 5 μl 10X ThermoPol Buffer
• 1 μl 10 mM dNTPs
• 2 μl 10 µM epPCR forward primer
• 2 μl 10 µM epPCR reverse primer
• 0.2 μl template DNA
• 0.5 μl Taq DNA polymerase without proofreading activity
• 34.3 μl ddH2O
• 5 μl 0.1-0.8 mM MnCl2

STEP	TEMP	TIME
Initial Denaturation	95 °C	60 sec
30 Cycles	95 °C	30 sec
	54 °C , determined using NEB Tm calculator	30 sec
	68 °C	60 sec
Final Extension	68 °C	2 minutes
Hold	4 °C

Gel purify PCR product and gibson assemble into backbone.

Optional: Add 1 μl DpnI enzyme to digest methylated DNA (background)

Work on ice.

• 1 μl (50-100ng) digested backbone DNA
• 2 μl (2-3x excess) of each DNA fragment

Add ddH2O to reach 5 μl, then add 5 μl 2x Gibson Assembly® Master Mix by NEB.

Incubate at 50°C for 1h, then transform or store at -20°C.

Work on ice.

• 1 μl (50 ng) digested backbone DNA
• 2 μl (37.5 ng) of each DNA fragment
• 1 μl 10x T4 DNA Ligase Buffer by NEB

Add ddH2O to reach 9 μl, then add 1 μl T4 DNA ligase by NEB.

Incubate at RT for 15 min or 1 h, then transform or store at -20°C.

1. Calculate volume of overnight culture needed for dilution to OD₆₀₀=1 in 3.5 ml final volume.
2. Transfer calculated amount into a falcon tube.
3. Centrifuge at 4500 x g for 5 min and resuspend in 3.5 ml PBS.
4. Dilute H₂O₂ stock solution (10 M) to the following concentrations: 0.01 mM, 0.1 mM, 1 mM, 2 mM, 5 mM, 10 mM.
5. Prepare a clear bottom black walls 96 well plate (Greiner 655097) and add 180 μl of resuspended cells + 20 μl H2O2 H₂O₂ per well. The final concentration in the well will be 10x lower than that of the prediluted H₂O₂ solution.
6. Load plate into TECAN plate reader and start the following program.

Before first use, add 40 μl of DMSO to the vial of DCFDA and mix well. Store at -20°C in the dark.

1. Prepare Reaction Master Mix: To 1 ml Assay buffer, add 2 μL ROS + DMSO detection reagent stock solution.
2. Centrifuge the cells from overnight culture and resuspend the cell pellets in PBS to reach OD 0.001 in 90 μL (at 100,000 cells/well) in a 96-well plate.
3. Add 110 μL of H₂O₂ solution to the cells (Final concentrations: 0, 0.1, 1 mM).
4. Incubate and measure in TECAN for 1h min at 37°C (Ex 520, Em 605).
5. Centrifuge the plate at 2250 x g for 7 minutes with brake off.
6. In half the wells, remove supernatant and replace with assay medium and resuspend, resuspend other pellets.
7. Incubate in TECAN for 1h min at 37°C.
8. Measure fluorescence in TECAN (Ex 520, Em 605).
9. Centrifuge the plate at 2250 x g for 7 minutes with brake off.
10. In half the wells, remove supernatant and replace with PBS and resuspend, resuspend other pellets.
11. Incubate in TECAN for 2h min at 37°C.
12. Centrifuge the plate at 2250 x g for 7 minutes with brake off.
13. Remove supernatant and replace with assay medium and resuspend and measure fluorescence in TECAN (Ex 520, Em 605).