Phenotypic Assortment to reduce Gene Copy Number

Materials

 * Tetrahymena stock cultures


 * SPP or NEFF medium


 * Selective Drug

Doubling Test

 * 1) Inoculate 2 mL SPP or NEFF medium in a 24-well plate with 100 μL of fresh stock culture. Add selective drug to working concentration. For example, in SPP the working concentration of paromomycin (pm) is 80 µg/mL which is what we would add to the SPP medium.
 * 2) Incubate overnight at 30 C.
 * 3) Inoculate 1 mL SPP or NEFF (use medium that is used previously) in a 24-well plate again with 100 μL of overnight culture. Double the working concentration of the selective drug and incubate overnight at 30 C again. Working with the example from above, doubling the concentration of pm in SPP would require us to use 160 µg/mL on day 2. 
 * 4) Passage cells cultures daily into media with doubled pm concentration until the culture fails to double in 24 hours.   Record OD540 from Day 1 and use that as standard for OD measurements for the next day. For example, if OD540 of cells was 0.12 on Day 1, then cells are assumed to have doubled if OD is >= 0.24 on Day 2. If cells are at OD < 0.24, then proceed to next step. 

Intermediate Drug Test

 * 1) Inoculate 2 mL SPP or NEFF medium in a 24-well plate with 100 μL of fresh culture. Increase the final drug concentration in increments of 50 µg/mL (or another intermediate drug concentration) and incubate overnight at 30 C.  Record pm conc. at which cells were last able to double in 24 hours. Increase the total pm concentration by increments of 50 µg/mL. For example, if the pm conc. at which the cells were last able to double was 500 µg/mL, then the next intermediate increase would be 500 + 50 = 550 µg/mL. And after that 600 µg/mL and so on and so forth. 
 * 2) Passage cells cultures daily into media with increments of drug concentration until the culture fails to double in 24 hours. This final concentration is the maximum drug concentration permissive for doubling in 24 hours.

Maintaining Growth at Maximum Permissive Drug Concentration

 * 1) Passage cultures daily at maximum drug concentration permissive (for doubling in 24 hours) for 4-6 weeks.

Establish Colonial Cell Lines

 * 1) Isolate single cells in drop medium to establish independent clonal cultures. Allow cells to grow overnight at 30 C.
 * 2) Select the best growing colonial cell line.

Testing Gene Essentiality

 * 1) To test whether a gene is essential, cells are released from selective drug selection and passaged daily in growth medium without drug for 2 weeks.  This allows an essential gene to back-assort from the limiting, possibly very low copy number present at maximal selection to a higher copy number optimal for growth and more readily detected by PCR or Southern blot analysis. 
 * 2) Test retention of gene or reduction in gene copy number by PCR or Soutehrn Blotting. Select final line for long-term storage.