Construction of Pf-5 mutants and derivative strains

Protocol for making a deletion of an interested gene in the chromosome of strain Pf-5

Day 1: Streak mutant Pf-5 strain on KB plate (no antibiotics) and grow overnight at 28°C.

Streak S17-1 E. coli strain carrying deletion construct carried by pEX18Tc to be introduced on LB + Tc₂₀ plate and grow overnight at 37°C.

Day 2: Start 4 mL KB culture of Pf-5 strain and shake overnight at 28°C.  Start a 4 mL LB + Tc₁₀ culture of S17-1 strain and shake overnight at 37°C. (Note: The S17-1 strains tend to grow slowly.  If this is a problem, try starting cultures in a.m. to allow 24 hrs to grow.)

Day 3: Next morning, check OD₆₀₀ of overnight cultures (1/10 dilution).

Spin down 1 mL Pf-5 strain and enough S17-1 strain to equal 1:1 ratio with Pf-5 based on OD₆₀₀.  (If S17-1 culture has very low OD, can use less Pf-5 to keep ratio 1:1).

Spin at 10,000 rpm for 1 min. 

Discard supernatant, resuspend in 1 mL LB, and spin again (10,000 rpm for 1 min).

Resuspend each pellet in 100 µL LB, mix two strains together and pipet (200 µL) onto a NA + glycerol plate in a pool (do not spread). Incubate 6 hrs at 37°C (right-side up). 

Add 4 mL 0.1 M MgSO₄ or sterilized water to plate and use sterile spreader to resuspend cells.  Pipet off solution containing cells into a sterile tube. Plate 100 µL undiluted and 1/10 dilution of mating mix on KB + Tc₂₀₀Sm₁₀₀.  Place in 28°C incubator.

Day 4: Mating plates may have a lawn of tiny colonies with larger colonies on top.  Colonies may not be visible until p.m.  Re-streak 6 of the large colonies (or less if you don’t get 6) on KB + Tc₂₀₀Sm₁₀₀.  If colonies too small to re-streak 1^st day, can let grow another day.

(Note:  Can make glycerol stocks after this step if can’t continue on right away.)

Day 5: Sucrose selection:  Use a toothpick to take a small “glob” of bacteria from each re-streaked bacterial colony and use to inoculate 1 mL LB (no antibiotics) in a microfuge tube.  Tape tubes on 28°C shaker for 2-3 hrs.  Make 1/10 and 1/100 dilutions and plate 100 µL of each on LB + 5% sucrose.  (Or plate 10 µL undiluted and 1/10 dilution on same plate.) Place in 28°C incubator.

Day 6:  Pick 24 colonies from plates that have well-separated colonies and re-streak on LB + 5% sucrose for single colonies (to ensure colonies are not mixed). Also patch onto KB + Tc₂₀₀.

Day 7: All colonies should grow on LB + 5% sucrose.  Only bacteria that retain the plasmid used to introduce the construct will grow on KB + Tc₂₀₀.  You want the clones that DO NOT GROW on KB + Tc₂₀₀ (resolved merodiploids will no longer carry Tet^R gene). Give them a full 24 hrs growth before confirming as Tet sensitive because sometimes slow growth occurs. (Should also check Tet plate at 48 hr to be sure no growth occurred.)

Pick single colony off LB + 5% sucrose plates from each of 12 Tet sensitive strains and patch onto LB + 5% sucrose or KB.

(Note:  Most of bacteria that grow on LB + 5% sucrose plate should not grow on KB + Tc200. However, sometimes you may find the bacteria can grow on both LB + 5% sucrose and KB + Tc200 plates, which indicates that the sacB gene is not functional in sucrose selection. If this is a problem, you may need to remake the deletion construct by using a confirmed vector)

Day 8: Confirm the deletion by PCR. Generally, you may initially make DNA (cell lysis) from 6 strains and perform PCR to test for the deletion.  If no mutant strains are identified, the other 6 clones can be tested.  12 strains are usually sufficient to identify a mutant, but some mutations may be more difficult to obtain, so you may want to have more clones as candidates. Once a mutant has been confirmed, streak on SMA (Skim milk agar) to ensure that it does not contain a gac mutation. At the same time, store the PCR positive mutants as glycerol stocks at -80°C. 

Use the colonies that show positive results from both PCR analysis and SMA assays for the next step research.