Creating Lac- E. coli Mutants

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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?


Day 1

Plate strain S-401 (Lac+, uvr phr (-)) from the -80 freezer onto a MacConkey or LB agar plate. Incubate at 37 degrees.

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

1. Prepare solution of bacteria and sterile PBS (depending on date, the O.D might differ) in test tube with green cap

a. 1.5 mL bacteria + 1.5 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 OD is ~0.4

5. If OD is not ~0.4, remove 1 mL of the solution and add 1 mL of either PBS or bacteria depending on whether OD is higher or lower than 0.4. Repeat until OD is ~0.4.

Undiluted bacteria to put in UV Stratalinker

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

2. Label one plate: “800 uJ’

3. Pipette 750 uL of bacteria (x 2) into plate (1.5 ml total)

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

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

5. Expose the bacteria to 800 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

Preparing MacConkey agar plates

1. Get 3 MacConkey agar plates for each of the following dilutions (so 9 plates in total)

a. 800 uJ, 100
b. 800 uJ, 10-1
c. 800 uJ, 10-2

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

Preparing Tubes

1. Label two micro centrifuge tubes according to their radiation exposure amount and dilution amount:

a. 800 uJ, 10-1
b. 800 uJ, 10-2

2. Place tubes in micro centrifuge tray (found near the beads and pipette boxes)

3. Fill each tube with 900 uL using sterile PBS bottle with pump (0.9 mL setting)

a. Move tubes to mark your progress


1. Use filter tips

2. Pipette 100 uL of bacteria from 800 uJ small plate and put into tube labelled “800 uJ, 10-1”

3. Vortex tube

4. Then pipette 100 uL of bacteria from “800 uJ, 10-1” tube into “800 uJ, 10-2” tube

5. Vortex “800 uJ, 10-2” tube

Final Steps

1. Pipette 100 uL from each 800 uJ micro centrifuge tube (10-1 and 10-2) into corresponding labeled agar plates

2. Pipette 100 uL from 800 uJ small plate directly into 800 uJ, 100 agar plates

3. 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

4. Flip agar plates so that they are upside down

a. Lid at the bottom

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

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

7. Place agar plates into incubator at 37 degrees C

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