sTAUbility

Protocols

POC Evolution Experiment Protocol

(Pre-made SD-complete, SD-EMS, SD-leu, SD-ura, 40% Glycerol)

Figure 1. Schematic diagram of plate 1 or 2.

Figure 2. Schematic diagram of plate 3 or 4.

Inside a biological hood Take a copy of the SWAT GFP/RFP library and pick the right strains (10 selected strains + 3 negative control + 2 WT control) using a tip according to the following map (SWAT excel sheet). Mix in a 96 well-plate (sterile with a lid) with 200 uL ddw.
Add 450uL SD-complete to sterile 96 deep-well plate “1”. Add 450uL SD-Complete-EMS to sterile 96 deep-well plate “2”.
Transfer 50 uL of diluted strains to the 96 deep-well plates “1”+”2” in the same order. Put on the plates a sterile breathable sticker and Transfer to ON incubation (30 celsius degrees).
Add 450uL SD-ura (for RFP) and SD-leu (for GFP) to sterile 96 deep-well plate “3”. Add 450uL SD-ura-EMS (for RFP) and SD-leu-EMS (for GFP) to sterile 96 deep-well plate “4” as a control for contamination.
Transfer 50 uL of diluted strains to the 96 deep-well plates “3”+”4” in the same order. Put on the plates a sterile breathable sticker and Transfer to ON incubation (30 celsius degrees).
Transfer 150uL of 40% Glycerol to a 96 well plate (sterile with a lid). Then add 50uL of each strain in the right order.
Transfer to -80 celsius degrees freezer.
On the following day take the 96 deep-well plates (1+2, 3+4) to a biological hood. From the plates 1+2 take 100uL to a new sterile 96 well-plates and centrifuge for 1 minute at 4000g.
Dispose the supernatant carefully and add 100uL PBS. pipette gently and transfer 100uL to a black 96 well-plate for fluorescence emission test in a plate reader.
Transfer 100uL from plates 3+4 to a new 96 well-plates for OD test (600nm) in a plate reader.
Transfer 50uL from plates 1+2 to a sterile 96 deep-well plate with 200 uL ddw in each well for dilution.
Repeat the steps 2-7 with new plates (1+2 and 3+4)

Gene-SEQ preliminary experiment protocol (Chi.Bio)

(Pre-made SD-complete, SD-EMS, pre-autoclave silicon tubes, 3 way valves, 25mL glass test tubes with stir bar and 3D-printed caps with nozzles)

Inside a biological hood transfer to the glass test tubes, 18mL of SD-complete and add 2mL of co-culture yeast mix starter(SWAT GFP/RFP library) that grew overnight and diluted to OD of 0.1. Put on the 3D-printed cap with the nozzles (figure 3).

Figure 3. 3D-printed cap with nozzles.

Transfer the flasks to the chi-bio reactors (figure 4) and transfer the silicon tubes to the chi-bio pump set by removing the screws on top of each pump and reattaching them afterward (figure 5).

Figure 4. The chi-bio reactor.

Figure 5. The chi-bio pump set.

Attach the silicon tubes to the designated nozzles of the glass test tube caps and on the designated nozzles of the media bottle 3D-printed GL-45 cap (figure 6) (SD-complete or SD-EMS). In addition, attach an air filter to all of the caps designated nozzles.

Figure 6. 3D-printed GL-45 cap with nozzles.

You may use a 3 way valve in parallel in order to distribute media from one bottle to multiple glass test tubes.

Open the web-based software and add the following parameters:

Desired OD set-point (0.8)
Desired test tube liquid level (20 mL)
Desired fluorescence wavelength (GFP/RFP)
Desired temperature (30 celsius degrees)

Calibrate the reactors according to chi-bio protocol (Link) and start the experiment.

Check the experiment from time to time and add fresh media if needed.

Restriction Endonuclease Reactions

Restriction Enzyme	10 units is sufficient, generally 1 uL is used
DNA	1 ug
10X NEBuffer	5 uL (1X)
Total Reaction Volume	50 uL
Incubation Time	1 hour*
Incubation Temperature	Enzyme dependent

Ligation Protocol with T4 DNA Ligase

COMPONENT	20 uL REACTION
T4 DNA Ligase Buffer (10X)*	2 uL
Vector DNA (4kb)	50 ng (0.020 pmol)
Insert DNA (1kb)	37.5 ng (0.060 pmol)
Nuclease-free water	To 20 uL
T4 DNA Ligase	1 uL

* The T4 DNA Ligase Buffer should be thawed and resuspended at room temperature.

Gently mix the reaction by pipetting up and down microfuge briefly.
For cohesive (sticky) ends, incubate at 16°C overnight or room temperature for 10 minutes.
For Blunt ends or single base overhangs, incubate at 16°C overnight or room temperature for 2 hours (alternatively, high concentration T4 DNA Ligase can be used in a 10 minute ligation).
Heat inactivate at 65°C for 10 minutes.
Chill on ice and transform 1-5 uL of the reaction into 50 uL competent cells.

Transformation to Yeast

Reference: Gietz, R.D. and R.A. Woods. (2002) TRANSFORMATION OF YEAST BY THE Liac/SS CARRIER DNA/PEG METHOD. Methods in Enzymology 350: 87-96.,

or, BETTER, his website: Link

All solutions are listed below. Note that there is also an OLD TRANSFORMATION METHOD that is as good as this for most purposes (see below).

This protocol can be used to generate sufficient transformants in a single reaction to screen multiple yeast genome equivalents for plasmids that complement a specific mutation. It can also be used to transform integrating plasmids, DNA fragments and oligonucleotides for yeast genome manipulation. The High Efficiency Protocol can also be employed to transform a yeast strain simultaneously with two different plasmids, such as the two-hybrid bait and prey plasmids.

REMEMBER!! EVERYTHING HAS TO REMAIN STERILE!! WORK NEAR A BUNZEN BURNER!!! NEVER LEAVE ANY BOTTLE/FLASK/PLATE OPEN!!!

Day -1

Inoculate the yeast strain into 5 ml of liquid medium (YPD or SC selection medium) and incubate overnight on a rotary shaker or roller at 30°C. ALWAYS prepare more than one starter, make sure you work in STERILE conditions.

Prepare 100 ml YPD in an Erlenmeyer of 500 ml or larger. If you have enough room, incubate the Erlenmayer at 30°C overnight.

Day 0

You will need your cells in the best physiological conditions. This is usually at mid-logarithmic growth (0.5-4x$10^{7}$cells/ml). To achieve the best possible cell population you can take the long road described. If you are in a hurry, see below “Alternative Day 0”.

Determine the titer of the yeast culture by pipetting 10 ml of cells into 1 ml of water in a spectrophotometer cuvette (1:100) and measuring the OD at 600 nm. For many yeast strains a suspension containing 1x$10^{6}$cells/ml will give an OD600 of 0.1. Alternatively, titer the culture using a hemocytometer (recommended; watch out for contaminating bacteria that float and move around!):

Dilute the overnight culture in water (100 ml of cells into 900 ml of water, 1:10).
Carefully place 10 µl of the cell suspension between the cover slip and the base of the haemocytometer. The grid area is typically 1 square millimeter, divided into 25 equal-sized squares, and the volume measured is $10^{-4}$ml.
Count the number of cells in 5 diagonal squares (we usually count the four corners and the central square).
Calculate the cell titer as follows: cells counted x 5 x dilution factor x $10^{4}$. For example, if you diluted your cells 1:10, and counted 203 cells in 5 squares, then it is: 203 (count) x 5 (to get the number in 25 squares) x 10 (dilution factor) x $10^{4}$ (volume) = 10,150 x $10^{4}$, that is 1.02 x $10^{8}$ cells/ml.
Saccharomyces cerevisiae divides by budding from a mother cell. Count small-budded cells as a single cell. Count cells with equal bud sizes as two cells when there is evidence of additional buds forming on either cell.

Transfer enough cells to the pre-warmed Erlenmayer with 100 ml YPD in order to get 0.5 x $10^{7}$ cells/ml (e.g.: 5 ml of your overnight culture above, when transferred to the 100 ml Erlenmayer, will be at the right concentration).
Incubate the flask on a rotary or reciprocating shaker at 30°C and 200 rpm.

Note:

For the best results, it is important to allow the cells to complete at least two divisions.
This will take 3 to 5 hours.
This culture will give sufficient cells for ~10 transformations.
Transformation efficiency (transformants/ µg plasmid/$10^{8}$ cells) remains constant for 3 to 4 cell divisions.

When the cell titer is at least 2 x $10^{7}$ cells/ml harvest the cells by centrifugation at 3000 g for 5 min, wash the cells in 25 ml of sterile water and resuspend in 1 ml of sterile water.

Alternative Day 0

You can organize your day so that the transformation starts first thing in the morning, by using one of the two following possibilities:

On Day 1, inoculate 100 ml YPD with enough cells so that at the time you want to start they will arrive to 2 x $10^{7}$ cells/ml [e.g., if at 5 pm the previous day you inoculate 100 ml YPD with 5 x 106 cells (5 x $10^{4}$/ml), by ~9 o’clock your cells will be at the right concentration].

An easy formula to make the calculation is:

Where n is the number of generations (obviously, the less generations, the more accurate)

Alternatively, use DIRECTLY the overnight starter, without diluting and growing the cells for 4 hs. This usually gives 5-fold lower efficiencies, which are usually good enough.

Boil a 1.0 ml sample of carrier DNA for 5 min and chill in an ice/water bath while harvesting the cells.

* It is not necessary or desirable to boil the carrier DNA every time. Keep a small aliquot in your own freezer box and boil after 3-4 freeze-thaws. But keep on ice when out.****

Transfer the cell suspension to a 1.5 ml microcentrifuge tube, centrifuge for 30 sec and discard the supernatant.
Add water to a final volume of 1.0 ml and vortex mix vigorously to resuspend the cells. You have at this point ~2 x $10^{9}$ cells in 1 ml.
Pipette 100 µl samples (ca. 108 cells) into 1.5 ml microfuge tubes, one for each transformation reaction, centrifuge at top speed for 30 sec and remove the supernatant.

IMPORTANT: Before you carry out the Trafo, remember Kupiec’s law #1: “In all transformation experiments, there will ALWAYS be a negative control (“no DNA”) and a positive control (a tube with 10-50 ng of a known episomal plasmid, to assess efficiency)”.

Make up sufficient Transformation Mix for the planned number of transformations plus one extra. Keep the Transformation Mix in ice/water.

Add 360 µl of Transformation Mix to each transformation tube and resuspend the cells by vortex mixing vigorously.
Incubate the tubes in a 42°C water bath for 20-40 min.

Note: The optimum time can vary for different yeast strains. Please test this if you need high efficiency from your transformations. For some strains, the longer the better (up to 3 hs), other strains die. In any case it is healthy to shake the cells by turning the tube down every half-hour.

Microcentrifuge at top speed for 30 sec and remove the Transformation Mix with a micropipette.
Pipette 1.0 ml of sterile water into each tube; stir the pellet gently with a micropipette tip. Don’t vortex.

Note: We like to be as gentle as possible at this step if high efficiency is important. Excessive washing washes away transformants!!!!

Plate appropriate dilutions of the cell suspension onto selective plates.

For transformation with an integrating plasmid (YIp), linear construct or oligonucleotide, plate 200 µl onto each of 5 plates; for a YEp, YRp or YCp library plasmids dilute 10 µl of the suspension into 1.0 ml of water and plate 10 and 100 µl samples onto two plates each. The 10-µl samples should be pipetted directly into 100 µl puddles of sterile water on the selective plates. Note that if you are transforming to resistance to drugs (G418, Hygromycin, etc.), you will need to resuspend the cells in YPD and allow at least one hour of incubation to allow expression prior to plating. Alternatively, plate on YPD, and the next day replicate the lane onto drug-containing plates.

Note: When spreading yeast cells onto the plate gently distribute the fluid completely with a sterile glass rod (“Drigalsky rod”) or glass beads with a minimum of strokes. TOO MUCH SHAKING KILLS THE CELLS. Allow the fluid to be taken up by the plate prior to incubation.

Incubate the plates at 30°C for 3 to 4 days and count the number of transformants.

The transformation efficiency (transformants/ 1 µg plasmid) depends on the type of plasmid used. Episomal plasmids (YpE, YpC and YpR plasmids) usually give >$10^{3}$ colonies/ 1 mg (Gietz claims efficiencies of up to $10^{6}$ colonies/mg, however, it is more common to get on the order of 104 colonies/mg). For integrative plasmids, the frequency is lower than 100 colonies/mg, unless they share homology with the genome; this increases the efficiency by 2-3 orders of magnitude.

Note that despite these numbers, transformation efficiency decreases with the amount of DNA used (Gietz et al. 1995). Gietz claims that the actual yield of transformants per transformation increases. For example, 100 nanogram of plasmid in a transformation might give a Transformation Efficiency of 5 x $10^{5}$ and a yield of 5 x $10^{4}$ actual transformants whereas with 1 µg of plasmid the Transformation Efficiency might be 2 x $10^{5}$ and the yield of actual transformants 2 x $10^{5}$. In order to obtain, lets say, 5 x $10^{5}$ transformants (total) it is simpler to set up two or three transformations with 1 µg of plasmid DNA, or a single 3 fold scaled up transformation, than to carry out 10 reactions with 100 ng of plasmid in each. However, this may not always be true, and sometimes 10 reactions with 100 ng are better (e.g., see his 2 Hybrid System TRAFO Page).

NOTE: Two plasmids, such as an expression plasmid and a library plasmid pool, can be co-transformed into a single cell by including both plasmids in the same transformation reaction. The efficiency is reduced, however, because only about 10% (Gietz claims 30-40 %) of all transformed cells take up more than one plasmid molecule. An alternative approach, which has a higher efficiency is to transform in the expression plasmid first, using this or the Quick and Easy protocol, and then use the protocol found on the 2 Hybrid System TRAFO Page to transform in the library plasmid pool.

NOTE ABOUT FREEZING: Competent cells can be frozen for future use. If you use one specific strain routinely, you may prepare the competent cells as explained, and at step 7, resuspend the cells in 30% glycerol or in Freezing Solution: [ 0.5 vol of 1.0 M. sorbitol, 10 mM Bicine-NaOH (pH 8.35), 3% ethylene glycol, 5% DMSO], then aliquote 100 ml cells/tube, and freeze at -70°C. When needed, just thaw the cells, spin down and transform as written above. This protocol, however, results in an order of magnitude lower transformation efficiencies.

Materials:

Single-stranded Carrier DNA (2 mg/ml): (No sonication is required anymore, bigger is better)

Weigh out 200 mgs of high molecular weight DNA (Deoxyribonucleic acid Sodium Salt Type III from Salmon Testes, Sigma D1626) into 100 ml of TE buffer (10 mM Tris-HCl pH 8.0, 1.0 mM EDTA). Disperse the DNA into solution by drawing it up and down repeatedly in a 10 ml pipet. Mix vigorously on a magnetic stirrer for 2-3 hours or until fully dissolved. If convenient, leave the covered solution mixing at this stage overnight in a cold room.
Aliquot the DNA and store in a -20°C freezer.
Prior to use, an aliquot should be placed in a boiling water bath for at least 5 min and quickly cooled in an ice water slurry.

TIPS:

Carrier DNA can be frozen after boiling and used 3 or 4 times. If transformation efficiencies begin to decrease with a batch of boiled carrier DNA it should be boiled again or a new aliquot used.
The lower concentration of carrier DNA (2 mg/ml) in this protocol eases handling and gives more reproducible results.
In previous protocol versions, a phenol: chloroform extraction was used to ensure maximal transformation efficiencies. This extraction may not be necessary if the DNA is of high enough quality. Test your carrier DNA to determine if extraction is necessary.
Note on Carrier DNA sterility: We dissolve our carrier DNA into sterile TE and then consider it sterile after the 5 min in a boiling water bath. Have not had problems with this approach!

1.0 M Lithium Acetate Stock Solution

The lithium acetate solution is prepared as a 1.0 M stock in distilled deionized water (dd water) and filter-sterilized or autoclaved. There is no need to titrate this solution, yet the final pH should be between 8.4 - 8.9.

Polyethylene glycol (PEG 50% w/v)

The polyethylene glycol (PEG), MW 3350 (Sigma P3640) is made up to 50% (w/v) with dd water and filter sterilized. For optimal transformation efficiencies, care must be taken to ensure that the PEG solution is at the proper concentration. In addition, it is important to store the PEG in a tightly capped container to prevent evaporation of water and a subsequent increase in PEG concentration. Small variations above or below the PEG concentration optimum in the transformation reaction, which is 33% (w/v), can reduce the production of transformants.

Place 50 gm of polyethylene glycol, MW 3350 (Sigma) in a 150 ml glass beaker and add 35 ml of ddH20.
Stir with a magnetic stirring bar until dissolved. This will take about 30 min.
Transfer all of the liquid to a 100 ml graduated cylinder. Rinse the beaker with a small amount of distilled water, add this to the graduated cylinder containing the PEG solution, and bring the volume to exactly 100 ml. Mix well by inversion.
Filter sterilize using a 0.45 µm filter unit (Nalgene) or autoclave, and store in a securely capped bottle.

TIPS:

Evaporation of the water from the PEG stock solution will result in an increase in the effective concentration of PEG in the transformation reaction and severely reduce the efficiency.

Plasmid DNA

Plasmid DNA can be prepared by standard protocols; extensive purification is not necessary for yeast transformation. RNA is an effective carrier in the LiAc/SS-DNA/PEG transformation procedure (Schiestl and Gietz, 1989) and does not need to be removed from plasmid DNA preparations before Transformation.

FACS Prep (Tyedmers Supplementary Protocol)

Yeast Culturing

Equipment:

Plate shaker (30°C)
Spectrophotometer capable of measuring optical density (OD) at 600nm
10 x 4 x 45mm disposable polystyrene cuvettes
15ml falcons
12 x 75 polypropylene tubes
Centrifuge at room temperature (20°C).

Reagents:

Yeast rich media (YPD)
Yeast selection media (SD-ura)
Phosphate-buffered saline (PBS)

Procedure

Strains preparation:

TIMING - 18 hours

Grow cells expressing the fluorescently tagged protein substrate of interest, in a pre-culture of selective/non selective liquid media (≤ 20ml) for 16-18hrs at 30°C with shaking (150 rpm).
Measure OD of cell suspension and dilute to an OD of 0.2 in fresh media (≤ 20ml).
Continue to culture cells at 30°C with shaking until a logarithmic growth phase is reached; OD 0.3-0.8 (ca.3hrs).

CRITICAL STEP: Do not let cells grow above an OD of 1.0; aggregation of the protein substrate will be affected when cells reach a saturated growth phase.

Sample preparation: Analysis with living cells.

TIMING - non-fixed cells 15 minutes

Transfer 5ml of the logarithmic culture into a 15ml tube and centrifuge at 4000g for 1 minute at room temperature.
Aspirate and discard the supernatant and gently resuspend in 1ml of PBS
Transfer the cell suspension to an appropriately labelled tube and store at room temperature, protected from the light.

CRITICAL STEP: Cells should be analysed within 1hr after collection of the cells.

Analysis of Samples by Flow cytometry

Equipment:

Flow cytometer, equipped with 488nm laser for excitation and appropriate filters for GFP/FITC); BD Canto or an equivalent
FlowJo version xV0.7 (OR, USA).
Graph pad Prism 6.05 (GraphPad Software, San Diego California USA)

Setup of Flow Cytometer & Running of Samples:

TIMING - 30 minutes

Ensure that the flow cytometer is equipped with the appropriate laser and filter sets to measure the fluorescently tagged protein of interest. This can be determined under the default cytometer configuration on the system. In the case of GFP, this would be 488nm laser for excitation and FITC/GFP channel for detection.
The flow cytometer must be able to collect the height, area and weight parameters for the channel of interest. For the BD Canto, these parameters can be selected under the Inspector – Cytometer menu. The GFP signal is best detected with logarithmic amplification
Select an appropriate flow rate for the samples. This parameter can vary depending upon the system used and should be determined empirically. For the BD Canto, a low (10µl/min) flow rate was selected.
Select the total number of events/cells to be collected per sample. The minimum number of events should be 50,000.
Create the following scatter plots under global worksheet:

Side scatter (SSC-A) versus forward scatter (FSC-A)
GFP-A versus FSC-A
GFP-H versus GFP-W

Create a statistical view that will display the mean fluorescent intensity (MFI) for the selected GFP parameters.
Set the voltage of the FSC-A and SSC-A channels, such that the smallest cells fit into the lower left 10% of the scatter plot. No forward scatter threshold is required, since all events from the culture were confirmed by microscopy to be intact and viable cells.
Determine the background fluorescence and minimum sample fluorescence. To set the photomultipliers (PMTs) of the fluorochrome of interest, a positive control exhibiting the fluorescence of choice and a non-fluorescent negative control are required. Using the GFP-A vs. FSC-A scatter plot the voltage of the GFP channel is adjusted so that the cells of the negative control fall within the lower quarter of the scatter plot (≤ $10^{3}$ GFP-A). A rectangular gate (P1) is then created above this region to define those cells that are GFP positive. This P1 gate is referred to as the parent and all cells t
Once the flow cytometer has been set up, as described above, the samples can be run. Mean running time per sample is ca. ≤ 10secs on BD Canto with the stated settings.
After the run, the samples can either be discarded or taken for further analysis by microscopy.

CRITICAL STEP: Yeast is a microorganism, it is therefore critical after running samples in the flow cytometer that a thorough cleaning of the system is performed to ensure that there is no residual sample left in the system leading to contamination of the fluidics. This is particularly relevant when working with live yeast. Users should discuss the best strategies for cleaning the head of flow cytometer core facility.

EMS Calibration

Pick up a single colony of 4741 strain (same genetic background as our GFP/RFP libraries) and mix it with 100uL of ddw.
Insert 450uL of SD-Complete to 10 wells in a 96 deep-well plate.
Add 50uL from the diluted strain to 9 of the 10 wells. (9 is negative control- no EMS, 10 is without yeast to make blank for the plate reader)
Add different amounts (table) of liquid EMS (diluted 1:10 in ddw) to each well.

Well number	1	2	3	4	5	6	7	8
EMS (uL)	1	5	10	25	50	75	100	200

Incubate ON (30 degrees).
Measure OD in plate-reader.
Centrifuge 1 min in 4000RPM. Discard supernatant ,Wash in ddw.
Add 100 uL of ddw and mix well.
Plate 100 uL of each well in Can+ plates.
Wait for 3 days
Count colony numbers on the plates.
Choose the EMS concentration that makes as many colonies on the Can+ plates, and less growth defects as measured by the plate reader.

PCR clean up

Purification:

ROCHE's PCR cleanup kit protocol

Preparation:

Take eppendorfs and write relevant names on them
Add 2µl* (the amount is for this experiment) of primer- reverse or forward (not both)
Add DNA after purification- the amount is according to its strength on PCR, for example: if it is strong add 2µl of DNA
Complete to 15µl with DDW
Pipetation
Put at -22°C

SD Media Preparation

Distilled water
Glucose Anhydrous
Yeast Nitrogen Base w/o Amino Acids
Amino acids (aa) mixture (according to proportion table). All aa from the list for SD-complete, for selection media, don’t add the specific aa to the mixture.

For 1 liter of media:

Add 900ml of ddw (double distilled water) ) to a glass bottle.
Add 20g of glucose.
Add 7.5g of nitrogen base.
Add 1.5g amino acid mixture.
Complete to final volume of 1 liter with ddw.

Team:TAU Israel/Protocols