competition-in-vitro.md

Competition in vitro

Competition of a V. fischeri mutant library through 15 generations in selective medium

1. Remove the glycerol stock from the -80 °C freezer. Work quickly so it does not thaw.

2. Using a sterile pipet tip, scrape a small aliquot of the stock (>25 μl) into a sterile microfuge tube on ice. Replace the original stock at -80 °C and allow the scraping to thaw.

3. Subculture 25 μl of the scraping into 2 ml LBS (1:80 subculture) and grow at 25 °C with aeration until the culture reaches an OD₆₀₀ of approximately 0.3. Record the OD.

4. Subculture 16.5 μl of the culture into 3 ml of the selective medium (1:181 subculture) and grow until the OD reaches the original OD. Note that 2^7.5 = 181, so the culture has now grown approximately 7.5 generations (doublings) in the selective medium. At this point freeze down an aliquot as a "frozen fossil record" and also proceed with the next step to grow the next 7.5 generations.

5. Subculture 16.5 μl of the culture from above and proceed to grow as above for the next 7.5 generations in selective medium. Note: if inoculating from a glycerol stock, use 24.75 μl to account for the glycerol volume. Once the OD returns, freeze the glycerol stock as before.

Notes:

• If growing for essential genes and the selective medium is LBS, then note that the strains get an extra ~ 6 generations in the LBS pregrowth. We have typically kept this pregrowth the same.
• What if the growth rates differ? This has to be examined biologically and is best examined with relevant control conditions.