Edited June 25, 2019

Library Generation (for SATAY 2.0 using pBK549) on solid medium

A lot of the tips for most efficient transposition remain true but are not as important as they used to be in the original protocol (using pBK257), since the transposition efficiency has been drastically improved.

Before starting, please read the cautionary information about pBK549

Overview:

1-Transform your favorite strain with pBK549 (Gal-transposase/MiniDs/CEN URA).

2-O/N preculture of transformants in SD-URA 0.2% glucose 2% raffinose to saturation.

3-Concentrate cells.

4-Spread cells on transposition plates (SD-ADE 2% Galactose).

5-Wait ~15 days. Survey #clones per square cm appearing over time, using a stereomicroscope

6-Harvest.

7-Re-seed harvest in 2l SD-ADE 2% Glucose, 2 500 000 cells/ml – let grow O/N until saturation

8-Harvest – wash cells – aliquot – freeze pellets

In bold are important parameters

(In italic) are useful tips.

Notes before you start:

• We (Kornmann lab) have only worked so far with BY4741, BY4742 and W303 genetic backgrounds. Therefore, if you need to use another genetic background, we strongly advise that you perform a small-scale transposition first, with our strain as a positive control.
  • This protocol will reliably yield ~1000 clones per cm2 for the BY4741 background (meaning 1000X55cm2 =55000 clones/plate) and 300-600 clones per cm2 for the W303 background.
  • You want to aim for at least 2 million clones. Increasing the number of clones will increase the number of hits, although not proportionally. My first library contained 1.6 million clones and yield 250 000 independent hits. My second library contained 4.7 million clones and yield 350 000 independent hits. The overall sequencing profile of the two libraries was remarkably comparable.
  • Plating 50 plates will be sufficient if you are in the BY4741 background (that accounts for the plates you will have to trash due to mold contamination), but the more the merrier. 50 plates correspond to 50X200microl = 10ml of a culture at OD 39. This means you need at least 100ml of pre-culture.
  • If you compare two libraries (control and experiment), I would strongly advise that all media (plates and liquids) be the same for the control library and the experiment library. If you need to pour the plates in batches, then I would advice that you distribute the plates from the different batches equally between the control and the experiment library.
  • We advise to generate sub-libraries made of clones as independent as possible, to dilute possible effects of passenger mutations (unrelated to what you are screening for).
  • The success of the protocol relies on proper amounts of cells being plated or inoculated. These parameters are critical to ensure:
    • Efficient transposition for optimal coverage of the genome
    • That the transposed clones are well represented during the re-seeding step
  • The three steps at which the cell concentration is important are:
    • The preculture of the pBK549 transformants, which is done directly from the transformation plate.
    • The plating to induce the transposition (done from the previously mentioned pre-culture grown over night) ← This Is The Most Crucial Step For Efficient Transposition
    • The reseeding of the harvested library.

The number of cells and their distribution on the plate is important for transposition efficiency and to ensure the transposed clones are of homogeneous size. It is critical that the cells are put at the proper density before plating and also plated very homogeneously over the entire plate.

Plating too many cells inhibits the transposition. Plating too few decreases the number of clones. We are aiming for an optimum here.

The best way to estimate if the plating is satisfying is:

1- do a small scale experiment with different concentrations to see which works best in your hand (our spectrophotometers might say different things, so it is probably wise to test if you need to make some adjustments).

2- observe the cell density after plating under a 10X objective, and compare to the pictures added to this protocol. Those pictures illustrate the “do” and “don't” (see below, Day 1).

Reagents:

1-Plates:

• per clone: 1x SD-ADE, 1x SD-URA, 1x SD complete
• SD-ADE 2% galactose (25ml - 55cm²) - Plates can be kept for a few weeks at 4C. Prepare 1-2 L for ~40-80 plates (1000 ml/25 ml=40).

Recipe for 1L SD-ADE 2% galactose plates:

• Autoclave 1.7g Yeast Nitrogen Base (without Ammonium Sulfate), 5g Ammonium Sulfate in 300ml ddH2O
• Autoclave 20g Agar in 500 mL H20
• Combine both autoclaved solutions.
• Add 100ml of 10X AA-ADE
  • ⚠ Not all AA formulation work! The recipe that we use is here. The CSM formulation from forMedium also works well.
• Add 100ml 20% Galactose (previously autoclaved)
• Pour 25ml per plate. (← this is standard procedure by us. We don't know if that makes any difference but we imagine it could).

2-Solutions:

• SD-URA 0.2% glucose 2% raffinose (count 50ml for 20 transposition plates)
• SD-ADE 2% glucose (2L per experiment – more if library is over 5 million clones, see below)
• 1L ddH2O

Material:

• 30C shaker
• 30C incubator or room
• Sterile glass flasks (250mL + 5L)
• Sterile glass beads (~50 ml per experiment)
• 50ml falcon tubes or any conical tube to collect the cells and that can stand 1600x g.
• Dispenser with sterile 5ml tips.
• Sterile glass beaker (or anything sterile and large enough to keep the tip of the dispenser clean without having to remove it).
• Glass rake (I make mine out of a pasteur pipette)
• Sterile glass beaker to store the rake(s)
• Spectrophotometer

Protocol:

If necessary, delete ADE2 in your strain of interest (primer sequence available upon request), or engineer your mutation in ByK352.

The week before the experiment:

-Transform pBK549 into your strain(s) of interest, plate on SD-URA. Streak ~10-20 transformants on both -Ura and -Ade plates. This step verifies that the plasmid has not already recombined upon transformation in the cells (independent of transposase expression). Indeed, the plasmid, now containing a repair template is prone to spontaneously recombine (see pBK549). On the streaks, the transformed strains should grow well on -Ura, while only a few colonies should appear on -Ade (the expected products of spurious recombination events).

-Choose transformants that have the desired property (robust growth on -Ura, only few colonies on -Ade)

-Note: A streak corresponding to 1/8 of a plate and grown for 2 days at 30C will be enough to inoculate 30-60ml.

Day 0

• Put SD-ADE 2% galactose plates O/N in 30C room or leave them O/N at room temp in an open plastic sleeve. (I keep my plates in their own plastic sleeves and I leave the bags/plastic sleeves open to eliminate condensation. I store the plates in a cardboard box. I leave the box open but I tape the adjacent flaps together, so as to make a “wall” that protects the plates from our 30C room ventilation.)
• In the early afternoon, scrape the streak of your chosen transformant and put the cells in a small volume (10-50 ml) of medium. Measure the OD of the harvest and use it to inoculate a SD-URA 0.2% glucose 2% raffinose culture at an OD600=0.16 – 0.18 (the 0.2% glucose allows for a smooth diauxic transition from glucose to raffinose). For reference, OD 0.2~2500.000cells/ml. A streak corresponding to 1/8 of a plate and grown for 2 days at 30C will be enough to inoculate 30-60ml. Estimate that you will need 50ml saturated culture to spread 20plates. You can start independent precultures, plate them separately, then combine the harvested clones before reseed. If you chose to reseed the libraries independently, you will have the possibility to barcode the DNA and keep the sequences separate as well.
• Grow at 30C on shaker (140-160rpm) for 18-20 hours.
• Reserve sterile glass beads.
• Reserve a few 50mls falcon tubes.

Day 1

-Measure OD₆₀₀. Expect OD₆₀₀ ~3.5 after 18-20h (always dilute 10X to read OD above 1).

-optional but recommended: Take an aliquot of the O/N culture, dilute 25000X to 40000X and plate 200microl on one SD-complete, and one SD-URA plates so as to obtain ~350 colonies per plate. This is just to check that the cells are ok and that most contain the plasmid (in case transposition does not work well). It is not unusual to find out that up to 30% of the clones have become ura^-.)

-For OD₆₀₀=3 and up:

• concentrate the culture to OD₆₀₀=39 (*)
• plate 200microl per plate using 5-15 glass beads per plate (oddly we found that this is actually important not to use too many beads).

(*)For example, if OD₆₀₀= 3.9, then you need to concentrate 39/3.9=10X. Spin culture 5min at 600x g at RT, discard 90% of the volume, mix pellet with remaining sup, plate 200microl per plate.

If using 50 ml falcon tubes, it is ok to do 2 rounds of spin. In this case, remove entirely the first sup, add 50 ml culture, spin, remove 40 ml (i.e 80%) of sup, mix pellet with remaining sup and plate.

We found these conditions optimal for our spectrophotometer. You may want to figure what they are for your spectro.

Note:

• Always plate 200microl (as I say below, plating homogeneously is key)
• Adjust concentration fold so that the OD₆₀₀ after concentration is 39.
• Use a dispenser with a sterile tip, that will make your life much easier. While I am spreading the plates, I am keeping the tip of the dispenser in a sterile place (like a sterile glass beaker)
• Plate very homogeneously – The key is to achieve a thin layer of cells on the whole surface of the plate . You can observe the plating using a stereomicroscope or a 10X objective: about 80% of the plate should be covered. See pictures for an idea of what the ideal and non ideal plating look like. In the end, it is the density on the plate that matters, so the best is to refer to the picture and to do a test run to figure out the density that works best in your hands (again, our spectrophotometers may have slightly different readings)
• To achieve an homogeneous plating, I do as follow:
  • - Dispense 200 μl onto 10 plates (already containing the beads)- This is stack 1
  • - Spread the liquid onto the entire surface of the plate, using a gentle circular motion, making sure the beads remain on the plate. Stop when the liquid starts to penetrate the plates. At this point, the cells form an homogeneous film that is just starting to be absorbed.
• Repeat with another stack – This is stack 2
• Gently shake stack 1 using straight motions, 2-3 times in 4 directions. After this step, the liquid should be fully absorbed. The cells form a monolayer, that occupies 50-80% of the surface of the plate (observed under 10X magnification). By eye you can see patterns on the surface of the plate.
• Remove the beads immediately (don’t let them sit)
• ...

Example of "good" plate after cell spreading, as seen with a 10X long distance objective (200μl of a suspension at OD38 plated).

Example of "bad" plate after cell spreading (suspension at OD77).

Note: adjust the number of plates per stack, if necessary (If the liquid does not get absorbed fast enough, increase the number of plates per stack)

• Plate as many plates in total as possible. It is better to decide not to harvest all of the plates than to find out that you don't have enough to achieve a good coverage. Take into account that ~10-15% might become moldy over the time that the transposition requires!!!
• Work as cleanly as possible to minimize moldy plates.
• Plate 200 μl on a few SD-ADE 2% glucose plates. This step controls for transposition having occurred before exposure to galactose. I would plate one SD-ADE plate per independent batch and I would label the SD-ADE plates according to their corresponding batches. ADE⁺ clones that have been generated before exposure to galactose will show on the SD-ADE plates and on the SD-ADE galactose after 2 days. This step is particularly important with the new protocol, as the plasmid with a repair template is highly recombinogenic. You will get a number of ADE+ cells on SD-Ade +glucose. You will have to ponder if this number is sufficiently low to not mess up the analysis. Bear in mind that you expect more than 50000 clones per plates on galactose, and therefore having 100-1000 Ade+ colonies on glucose is no big deal.
• Place the plates in closed plastic sleeves/bags (and then back in their card box). Leave an opening for air flow (do not seal tight to avoid growth in anaerobie and accumulation of condensation) but make sure the plates will not dry out.

Day 2-3

-Check plates: SD-ADE should have a reasonable number of colonies – Count SD complete and SD-URA plates.

-Check plates for mold! Remove moldy plates, wipe any plate preceding and following moldy plates in the stack with 70% EtOH.

Here is an approximate timing of what should happen next. No need to follow it precisely.

Day 6

-Check plates for mold! Remove moldy plates, wipe the preceding and the following plate with 70% EtOH. Do it again in the following days.

-Clones may start appearing already.

Day 10-12

-Librairies generated in the W303 background will be ready around day 12-13

-Clones should start appearing :) . They will be visible under the stereomicroscope and 10X objective. Don't panic if you don't see any, this is still very early.

Day 13-15

-You should start seeing the clones by eye. If all plates have equal clone density, pick 10 plates and count as below. If plates seem to have have unequal clone density, try to make categories and pick a few plates per category to proceed. This is important to get an approximate number of clones.

-Draw 1cm² on each selected plate. Count clones within this area using a stereomicroscope (and a permanent marker). Note the number and the date on the plate (see picture).

Note: We will add proper pictures when we have them

Day 16-18

-Count additional clones within the same areas as before (use a marker of a different color). Note number and date on the plate.

-BY4741-based libraries should be ready and it should become very tricky to count individual clones in the BY background because of their high density.

Day 20=day before the harvest

-Get a final count for your number of clones

Harvest day

-Prepare and pre-warm 2 liters of SD-ADE 2% glucose. A two-liter volume is a good compromise to make sure that 1. each independent clone is represented adequately by a large number of cells in the culture and 2. It remains easy to handle. You can calculate the representation of each individual clone by dividing the number of cells that you inoculate, by the number of clones that you estimate you have. Make sure this number is above 100.

-If you don't have a glass rake, make one by flaming a Pasteur pipette – Keep it in a sterile glass beaker.

-Proceed with batches of 5 plates (more if that works for you), as follow:

-Distributes 2 ml ddH2O per plate

-Scrape one plate (I like to keep the plate flat on the bench and rotate it), gathers the liquid to one “side” of the plate, and handles the plate to person 1 (keeping it tilted) or places the plate on a wedge.

-Then aspirate the liquid from all 5 plates into a sterile 250ml glass Erlenmeyer.

Here is a video illustrating the choreography with three people:

-Note that the above video was set for a harvest of 350 plates, which is a sizeable endeavour. Since 50 plates are now sufficient, the harvest can easily be performed by one single scientist in a reasonable amount of time.

-Make suitable dilution to measure the OD₆₀₀ of the collected cells and count cells with a cytometer.

-Inoculate the 2 liters at a final concentration of 2.5E6 cells/ml (that is OD=0.2 on our spectro). My advice is to inoculate less than what you have calculated, measure, then adjust.

-Proceed as above for every growth condition to be tested.

Day after harvest

• Collect at least 600ml of saturated culture (OD~4), 5min, 1600g, Room Temp. Do two rounds if necessary (If not limited by centrifuges, spin the whole 2 liters. You'll have more material to mess around).
• Wash the equivalent of 600ml culture with 150ml ddH2O (5min, RT, 1600x g).
• Weight the pellet(s). 600ml culture at OD600~4 should yield ~4 gram pellet.
• Resuspend the pellet in minimal amount of ddH2O,
• Distribute in microtubes so that each tube contains ~500mg packed cells.
• Spin (5min, 1600g or a few sec at 16000g), remove all of the sup, store at -20C.