GENE REGULATION: INDUCTION OF THE lac OPERON IN E. coli ©David B. Fankhauser, Ph.D. Professor of Biology and Chemistry University of Cincinnati Clermont College, Batavia OH 45103
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      This is an experiment to demonstrate that the lactose operon in E. coli is repressed when grown on glucose, but can be induced to produce lactose metabolizing enzymes when grown on lactose.  The experiment will consist of three steps:

Per group of four:
Label five tubes 13 x 100 mm  for time the aliquot was collected from the culture:
        T-0, T-20, T-40, T-60 and T-80. (# = minutes grown in lactose)

	1.	Grow E. coli B over night with shaking in 50 mL CSHA minimal medium + 0.1%.
	2.	Spin down cells in a 16 x 150 mm tubes, 10 mL each, in a clinical centrifuge at setting of 4 for 10 min.  Decant as much of liquid off as possible (remove glucose medium), save pellets.
	3.	Resuspend each pellet in 10 mL CSHA + 1% lactose.  Read A660.  Calculate how to prepare at least 25mL/team of four of a dilution of the cells in CSHA + 1% lactose to yield A660 of 0.200 in sterile plugged 250 mL flask.  (Show your math.)
	4.	Withdraw T-0, begin incubation with aeration: Transfer 7 mL for each team of four from the culture flask into a cuvette.  Place culture flask back in 37°C shaking incubator.  Start a timer to measure the time the culture has grown in lactose medium. Then immediately read and record the A660 of the T-0 aliquot, transfer to the 13x100 tube labeled T-0.  Immediately add a drop or two of toluene, cover with a double layer of parafilm, shake well, place on ice.
	5.	Withdraw T-20 after 20 minutes: transfer 7 mL per each team of four into a cuvette.  Read and record its A660, transfer to the 13x100 tube T-20.  Add a drop or two of toluene, cover with parafilm, shake well, place on ice.
	6.	Withdraw T-40 after 20 minutes more (40 minutes total): transfer 3 mL for each team of four into a cuvette.  Read and record its A660, transfer to the 13x100 tube T-40.  Add a drop or two of toluene, cover with parafilm, shake well, place on ice.
	7.	Withdraw T- 60 after 20 minutes more (60 minutes total): transfer 3 mL for each team of four into a cuvette.  Read and record its A660, transfer to the 13x100 tube T-60.  Add a drop or two of toluene, cover with parafilm, shake well, place on ice.
	8.	Withdraw T- 80 after 20 minutes more (80 minutes total): transfer 3 mL for each team of four into a cuvette.  Read and record its A660, transfer to the 13x100 tube T-60.  Add a drop or two of toluene, cover with parafilm, shake well, place on ice.
	9.	Perform assay of β galactosidase in each of the samples.     (See protocol β galactosidase assay in toluenized cells.)

  
   
  
  
  
 
1. Grow E. coli B over night (ON) with aeration in 5 mL CSHA minimal medium + 0.1% glucose in 15x150 mm tube.

2. Spin down cells in same tube in a clinical centrifuge at setting of 4 for 10 min. Decant as much of liquid off as possible (remove glucose medium).

3. Resuspend in 10 mL CSHA + 1% lactose. Read A_660. Calculate and add sufficient additional CSHA + 1% lactose to yield A₆₆₀ of 0.200 mL in sterile plugged 250 mL flask(final volume at least 50mL). (Show your math.) 

4. Withdraw T-0, begin incubation with aeration: Transfer 5 mL per each team of four from the culture flask into a cuvette. Place culture flask in shaking incubator, 37C. Start a timer to measure the time the culture has grown in lactose medium. Then immediately read and record the A₆₆₀ of the T-0 aliquot, transfer to the 13x100 tube labeled T-0. Add a drop or two of toluene, cover with a double layer of parafilm, shake well, place on ice.

5. Withdraw T-20 after 20 minutes: transfer 3 mL per each team of four into a cuvette. Read and record its A₆₆₀, transfer to the 13x100 tube C 20. Add a drop or two of toluene, cover with parafilm, shake well, place on ice.

6. Withdraw T-40 after 20 minutes more (40 minutes total): transfer 3 mL per each team of four into a cuvette. Read and record its A₆₆₀, transfer to the 13x100 tube T-40. Add a drop or two of toluene, cover with parafilm, shake well, place on ice.

7. Withdraw T- 60 after 20 minutes more (60 minutes total): transfer 3 mL per each team of four into a cuvette. Read and record its A₆₆₀, transfer to the 13x100 tube T-60. Add a drop or two of toluene, cover with parafilm, shake well, place on ice.

8. Perform assay of galactosidase in each of the samples.

(See protocolgalactosidase assay in toluenized cells.)

Your team should now have four samples of toluenized cells: 0, 20, 40 and 60 minute lactose-grown from the previous stage of the experiment. A preliminary experiment should have been run last week to determine repressed and derepressed levels of galactosidase and optimum assay conditions for these toluenized cells. You will now assay the amount of galactosidase in this set of cells using the same protocol.
 
 

1. Add 2 mL 2% K₂CO₃ to each K₂CO₃ tube with repeater pipet.

2. Prepare large batch of reaction mix in 25 mL graduated cylinder (20 mL total/4 students):

3.0 mL 0.1 M PO₄ buffer, pH 6.5
7.5 mL 20 mM ONPG
12.0 mL dH₂O

3. Assay the culture samples. Set up the following table in your notebook (2x spaced)
 

tube	sample	A660	dH2O	Rxn Mix	toluenized cells		A415	F - S	A415/A660
R-0	T-0		--	2.0	2.0	S
						F
R-20	T-20		1.0	2.0	1.0	S
						F
R-40	T-40		1.5	2.0	0.5	S
						F
R-60	T-60		1.5	2.0	0.5	S
						F

Set up tubes: add water, then 2 mL Rxn Mix to each of the tubes. Pre-incubate, 37C, 1min. For the four toluenized cell samples: start assaying T-0 first, staggering the starting times for each sample by 30 seconds. Perform the following operations for each sample:

a: Add specified volume of toluenized cells to appropriate assay tube: 2.0 mL for T-0 (repressed), 1.0 mL for T-20, 0.5 for T-40 and T-60, mix. (Final volume = 4.0 mL) Warm up for 1 minute 37C.

b: Withdraw 1.0 mL start sample from R-0 assay tube, add to the S-0 tube (contains K₂CO₃), mix, and start timing. Place sample in rack to be read later.

c: At 30 second intervals, withdraw start samples from R-20, R-40 and R-60.

• Incubate the four assay tube for 15 minutes at 37C.

• Read the A₄₁₅ of all tubes. For each culture sample, subtract the start from the finish = A₄₁₅ (which is proportional to the amount of enzyme present).

(1/mL of cells tested) x (A₄₁₅ from assay)/(A₆₆₀ of the culture at sampling time)

6. Graph the specific activities of the two cultures versus time. Discuss differences in specific activity seen as the culture grows for progressively longer times in lactose.