Liquid library generation W303

The following protocol has been optimized for the W303 genetic background. It doesn’t work quite as well in the BY4741 background. We do not know for any other backgrounds.

1-Transform pBK549 in W303

Streak individual transformants (6 per plate) on SD-URA and SD-ADE, grow for 2 days at 30C.

Pursue with clones that fully grow on SD-URA and form a few clones (~10) on SD-ADE (avoid those that fully grow on SD-ADE AND those that do not form any clone)

2-Preculture:

Scrape cells from the SD-URA plate, inoculate 20-30ml SD-URA 0.2% Glucose, 2% Raffinose at OD600= 0.16-0.24 (Note: I have tried 0.06, that works too) – Incubate at 30C, 140 to 160rpm, to OD600=4-6 (Count 20h from OD=0.23 to OD4 – 25h from OD 0.06 to OD6).

My advice is to start 3-4 precultures and later chose which to reseed in SD-ADE 2% Glucose

3-Induction:

Inoculate 200ml SD-URA 2% Galactose at OD600=0.2 from the saturated preculture- Incubate at 30C, 140 to 160rpm for 50-52 hours.

To evaluate the background:

-Spread 200 ul of this inoculum on SD-ADE - Dilute the inoculum 1000X, spread 200 ul on SD-URA and SD complete

Score after 2 days – Expect ~200-400 ADE+ clones/ml culture, i. e ~0.01%-0.02% of the total number of cells. These are cells that repaired the ADE2 gene by homologous recombination WITHOUT transposition.

-The OD should have reached 4-5 after 20 h induction– You want to have an idea of how well the transposition is going before reseeding. To do so, spread 200 ul directly on SD-ADE and spread 200 ul of a 40000X dilution on SD and on SD-URA. Score under a binocular after 30 h (i.e closer to the time of reseed). The number of clones you see is about 80% of the actual number - Expect 0.5-1% of the cells to have become ADE+. Incubate the plate one more day for a final count.

Pursue with the culture that gives the highest number of clones, starting from a minimum of background. 500-1000X more ADE+ clones at T51 than at T0 of induction is in the ballpark.

Note1: scoring after 20h will already give you ~40% of the actual # of ADE+ clones

Note2: exceeding 52 hours induction will not increase transposition efficiency. If anything, it might even lead to a loss of viability. In doubt, try a small scale time course: Set up the culture, measure OD at regular intervals, spread 200 ul aliquots on SD, SD-URA and SD-ADE. It takes about 17 hours to reach OD3. From then, dilute the culture 4000X to spread on SD and SD-URA. Transposition will already start between 0-20hours induction

Reseed:

Determine the number of ADE+ clones after 50-52 hours induction–2.3E5 ADE+ cells per ml is great. Inoculate 14 million cells in SD-ADE 2% Glucose at OD 0.2 – That should be about 2-4L – Incubate at 30C, 140rpm, harvest at OD~2 (~80hours). Note that the final OD will not exceed 2. For reasons we ignore, the presence of ade- cells in the mix inhibits the growth of ADE+ cells. This is why the culture will not grow above OD2. This is also why you should not reinoculate at an OD above 0.2.

To estimate the growth of ADE+ cells: dilute the culture 2000X, spread 200 ul on SD-ADE – dilute 40000X, spread 200 ul on SD and SD-URA – Expect the number of ADE+ cells to have been multiplied by ~1000X.

Note1: If you don’t mind increasing the volume, you can reseed at a lower OD, and thus have less growth inhibition. But in any case you want to inoculate enough cells so as to keep a good complexity in your library.

For example, a library containing 1.8E5 ADE+ cells/ml (OD=7.27, 0.15% ADE+ cells) after induction and reseeded at OD0.2 in 2L (1E7 independent transpositions) will reach an OD of 2 and a fold increase of 1250

– the same library reseeded at OD0.1 in 2L (5E6 independent transpositions) will reach OD3 for a fold increase of 3700

Harvest cells and proceed to DNA extraction as in published protocol.

Because of the growth inhibition, only ~70% of the harvested cells are ADE+, meaning that during the sequencing reaction, 30% of the reads will map to the untransposed transposon in ADE2.