Espress'EAU - Experiments

Protocols

General

Agarose Gel Preparation

Materials

50ml TAE buffer
0.5g Agarose
3μl Gel Stain

Procedure

mix TAE and agarose
microwave for 1.5 minutes
add gel stain
pour into gel tray and insert combs

cPCR validation

Materials

Phire Plant Direct PCR Master Mix (ThermoFisher)
Forward/Reverse primer (100µM)
H2O
Transformed Colonies

Procedure

Select and mark 10 colonies on the plate of interest
Prepare master mix according to the table below, and distribute into labeled PCR-tubes

Master mix for one PCR reaction
	Volume (µl)
H2O	12.25
Phire Plant Direct PCR Master Mix	12.5
Forward primer	0.125
Reverse primer	0.125
Total	25

Touch colonies and inoculate in PCR-tubes
Run PCR (33 cycles)

cPCR reaction cycle parameters. Must be run 33 times.
Temperature (ºC)	Time (minutes)
98	5:00
98	0:05
62	0:05
72	1:30
72	1:00
4	∞

Prepare a 1% agarose gel
Load PCR reactions on gel and run the gel for 25 minutes at 120 Volts
Take a UV image of the gel

Six parts assembly

Materials

Master mix for BsaI assembly of six parts:

Reagent	Concentration (ng/µl)	Volume(µl)
DNA Part 1	83	0.7
DNA Part 2	100	1.5
DNA Part 3	70.8	1.41
DNA Part 4	67.5	1.48
DNA Part 5	84.5	1.18
DNA Part 6	110	0.91
10x T4 ligase buffer		2
T4 ligase (NEB)		2
BsaI		1
dH2O		7.81
TOTAL:		20

DNA part 1: assembly connector [ConLS]

DNA part 2: 5 promoters [HSP12pr], [TRX2pr], [YCF1pr], [GSH1pr], [GLR1pr]

DNA Part 3: coding sequence [yomScarlet-i]

DNA Part 4: Terminator [tADH1]

DNA Part 5: Assembly connector [ConRE]

DNA Part 6: E.coli marker and origin [AmpR-ColE1]

Procedure

Mix:

add H2O
add 10x T4 ligase buffer
add BsaI
add T4 ligase (NEB)
add differents parts of DNA

Thermal cycling :

Cycle with the following parameters:
[42°C, 2 min.; 16°C 5 min.] x25
60°C, 10 min.
80°C, 20 min.
4°C, ∞

Two parts assembly

Materials

Reagent	Concentration (ng/µl)	Volume(µl)
DNA Part 1	100	1
DNA Part 2	200	0.5
10x T4 ligase buffer		2
T4 ligase (NEB)		1
BsaI		1
dH2O		14.5
TOTAL:		20

DNA part 1: promoter cassette

DNA part 2: Lys2-GFP dropout multi-gene backbone

Procedure

Mix:

add H2O
add 10x T4 ligase buffer
add BsmBI
add T4 ligase (NEB)
add differents parts of DNA

Thermal cycling :

Cycle with the following parameters:
[42°C, 2 min.; 16°C 5 min.] x25
60°C, 10 min.
80°C, 20 min.
4°C, ∞

Yeast

Yeast Transformation

The following protocol uses the LiAc/SS carrier DNA/PEG method for yeast transformation and is based on Gietz et al. (2007)¹.

Materials

Shaking incubator at 30°C
Stationary incubator at 30°C
Water bath at 42°C
Heat block at 105°C
SC-URA plate
5-FOA plate
Centrifuge
YPD medium
Lithium acetate dihydrate
PEG 3350
Integration cassette

Procedure

Day 0

Pick colony from plate into 5 ml growth media for overnight growth in 30°C shaker

Day 1

Turn on water bath to 42 °C.
Prepare an ice-water bath.
Set dry-block to 105 °C.
Place a SC -ura plate into the 30 °C incubator.

Cells preparation

Back dilute over night culture to OD 0.175 in 5 ml YPD.
Grow up yeast to OD600 ~0.75 at 30°C. Takes 4-5 hours for WT in YPD.
Pellet at 2000 rpm for 10 min in large centrifuge.
Prepare lithium acetate transformation while harvesting cells (see table).

Lithium Acetate mix for one transformation
	Volume (µl)
PEG-3350 (50% w/v)	260
LiOAc 1.0 M	36
ssDNA (10 mg/mL)	0.125
Total	306

Integration DNA prep
	Volume (µl)
Integration cassette	20
Cas9 (100ng)	5
Guide RNA (200 ng of each gRNA)	4
H2O	25
Total	54

Wash with 0.5x volume H2O and repeat step 8.
Wash with 0.5x volume 100 mM LiOAc and repeat step 8.
Final suspension in 0.01x volume 100 mM LiOAc (100 μL per transformation - approx 300 μl from 15 mL culture).
Pellet at 8000 rpm for 1 min at 20°C on small tabletop centrifuge.

Heat shock

Resuspend in the lithium acetate transformation mix. I suggest resuspending with the plasmid DNA and ddH2O (54 μL) first, then mixing with the LiOAc+PEG+ssDNA mixture (306 μL) afterwards, as it is too viscous to easily disrupt the pellet.
Incubate at 30°C for 30 min.
Incubate for another 17 min at 42°C in water bath (or heat block).
Centrifuge at 8000 rpm for 2 min and remove supernatant.
Depending on the selection marker: a. Prototrophic/auxotrophy gene: Resuspend in 200 μL of H2O. b. Eukaryotic antibiotic: Recover in 1.0 mL YPD for 2-3 hrs at 30°C.
Plate desired volume on plates. Typically do half.

Day 2

If there are colonies streak them on a 5-FOA plate for curing.

Pesticide Assay

The pesticide assay was carried out using a selection of the most frequently detected pesticides in groundwater (Based on the 2016 NAQUA Swiss groundwater monitoring report ²). Those pesticides were diluted over a range of concentrations to be tested (1000µl/L - 0.1µl/L). Yeast cells were grown with the presence of pesticides at 30°C.

Materials

Pesticides
Yeast strains (overnight cultures)
YPD medium
SC medium
96-well plate
Breathable membrane

Procedure

Serially dilute the pesticides in deionized water to the desired concentrations
Dilute yeast overnight cultures to 0.05 OD600 in YPD medium (only growth measurements), or to 0.3 OD600 in SC medium (fluorescence + growth measurements).
Pipette 99 µl of the cultures into the wells of the 96-well plate and add 1 µl of the pesticide dilution
Cover the plate with a breathable membrane to prevent evaporation
Insert in the plate reader and run for 18 to 24 hours while measuring the OD600 and the fluorescence (excitation wavelength = 570 nm, emission wavelength = 593 nm) over this period of time.

Bacteria

Bacterial Transformation

Materials

Shaking incubator at 37°C
Stationary incubator at 37°C
Water bath at 42°C
Ice bucket filled with ice
Microcentrifuge tubes
Glass beads
LB agar plate (with appropriate antibiotic)
SOC medium
LB medium
Competent cells
DNA (to transform)

Procedure

Add total volume of master mix (20.0 μL) to 50 μL chemically competent cells (e.g., BL21) in a 2.0mL tube.
Incubate on ice for 5 min. heat shock at 42°C for exactly 45 sec., immediately place on ice.
Add 950 μL sterile SOC medium.
Grow with shaking at 37°C for 30 min.
Pellet the cells at 4000g speed in a microcentrifuge for 3 min. at room temp.
Discard the supernatant. Resuspend the cells in 100 μL LB.
make a 100µl 10x dilution.
Plate the original and diluted cells on pre-warmed LB agar (with antibiotic) with sterile glass beads.
Grow overnight at 37°C.

DNA Extraction

Materials

Bacterial overnight cultures
P1 Buffer (Qiagen)
P2 Buffer (Qiagen)
N3 Buffer (Qiagen)
PE Buffer (Qiagen)
Microcentrifuge tubes
QIAprep 2.0 spin column

Procedure

Pellet 1–5 ml bacterial overnight culture by centrifugation at >8000 rpm (6800 x g) for 3 min at room temperature (15–25°C).
Resuspend pelleted bacterial cells in 250 μl Buffer P1 and transfer to a microcentrifuge tube.
Add 250 μl Buffer P2 and mix thoroughly by inverting the tube 4–6 times until the solution becomes clear. Do not allow the lysis reaction to proceed for more than 5 min. If using LyseBlue reagent, the solution will turn blue.
Add 350 μl Buffer N3 and mix immediately and thoroughly by inverting the tube 4–6 times. If using LyseBlue reagent, the solution will turn colorless.
Centrifuge for 10 min at 13,000 rpm (~17,900 x g) in a table-top microcentrifuge.
Apply 800 μl supernatant from step 5 to the QIAprep 2.0 spin column by pipetting. Centrifuge for 30–60 s and discard the flow-through
Wash the QIAprep 2.0 spin column by adding 0.75 ml Buffer PE. Centrifuge for 30–60 s and discard the flow-through.
Centrifuge for 1 min to remove residual wash buffer.
Place the QIAprep 2.0 column in a clean 1.5 ml microcentrifuge tube. To elute DNA, add 50 μl water to the center of the QIAprep 2.0 spin column, let stand for 1 min, and centrifuge for 1 min.