Creating Lac- E. coli Mutants

Goal

To create Lac negative E. coli mutants for student/faculty use.

Pre-Protocol Questions

1. Do you know how to use the spectrophotometer? The stratalinker?

2. Do you have enough PBS? Do you have enough bacteria?

Protocol

Day 1

Plate strain S-398 (strain W3110, Lac+) from the -80 freezer onto a MacConkey or LB agar plate. Incubate at 37 degrees C.

Day 2

Sterilely put 10 ml of LB into a sterile flask. Select one colony from the incubated plate, and inoculate the LB. Incubate in the shaking incubator at 37 degrees.

Day 3

Preparing Solutions

0. Turn heat block / waterbath to 47 degrees. 1. Prepare solution of bacteria and sterile PBS (depending on date, the Absorbance might differ) in test tube with green cap

a. 475ul bacteria from overnight culture + 4.5 mL sterile PBS

2. Use a blank containing at least 3 mL of PBS, insert into spectrophotometer, and press the arrow up button

3. Take out blank and insert test tube into the spectrophotometer

a. Spectrophotometer located near the iPads

4. Check if Absorbance is ~0.4 through using the solution of bacteria and PBS and a 13mm test tube.

5. If Absorbance is not ~0.4, remove 1 mL of the solution and add 1 mL of either PBS or bacteria depending on whether Absorbance is higher or lower than 0.4. Continue to adjust the absorbance until it is ~0.4.

Diluted bacteria to put in UV Stratalinker

1. Prepare a small petri plate (found in drawer near the area with plastic “to be autoclaved” bins)

2. Pipette 750 uL of bacteria that you have just diluted (Absorbance 0.4) twice into plate (1.5 ml total). Gently rotate the plate to create an even layer of bacteria across the entire surface of the small Petri dish.

3. Cover with lid and put into tray to carry to Stratalinker

a. Stratalinker located next to the balance/across from the pH meter

4. Expose the bacteria to 375 uJ of UV radiation

a. Make sure the “uJ” sign has a light next to it

b. Must remove the lid of each plate before placing it into the Stratalinker

c. Place plate at the center

5. Heat shock the bacteria to increase effective mutation rate

a. Pipette ~1.2ml bacteria into 1.5ml microcentrifuge tube

b. Incubate in heat block / waterbath at 47 degrees for 30 minutes

c. Immediately plate onto MacConkey agar

Plating onto MacConkey agar plates

1. Get 12 MacConkey agar plates; warm up in 37 degree incubator

2. Fill each plate with 4-6 beads to spread bacteria (beads are in large tubes overhead on lab table)

3. Vortex tube

4. Pipette 100 uL from heat-shocked bacteria onto the MacConkey agar plate.

5. Shake agar plates back and forth for approximately 30 seconds

a. Do not shake in a circular motion

b. Motion should preferably result in straight lines across the plates

6. Flip agar plates so that they are upside down

a. Lid at the bottom

7. Dispose of the beads into the white bead container (has a funnel on top).

8. Spray white bead container funnel with ethanol, to kill off any bacteria on it.

9. Place agar plates into incubator at 37 degrees C

Finding Mutants

1. Using a dissecting microscope, find white colonies among the pink colonies. In Eric's experience, slimy white colonies have a tendency to revert, so focus on 'normal' looking white colonies.

2. Circle any white colonies; screen colonies quickly, as there should be about 1000 colonies per plate.

3. Transfer colonies to a second MacConkey plate to ensure they are lac-. I plate 8-10 mutants per MacConkey plate to save on plates.

4. After overnight incubation, check for lac status. If still lac-, grow up cultures in liquid LB overnight and freeze down into the -80 freezer.

What Are We Looking For?

1. Bacteria that have been exposed to UV light should be mutated

2. Expect to see colonies that are completely white instead of being completely pinkish/purple

a. I.e., lac negative bacteria

3. Use a sharpie/marker to circle on the plates where these colonies are

4. Using sterile technique and the wire inoculating loop, transfer white colonies to a new MacConkey agar plate, to check that they are lac-. Incubate overnight.

5. Grow up an overnight culture of any lac- mutants in 5mL LB at 37 degrees in the shaking incubator

6. Talk to Eric about printing a label for these mutants; prepare to be frozen at -80 degrees