ASSAY OF GALACTOSIDASE IN INDUCED BACTERIAL CELLS page 35

March 1994, rvsd 27 Dec 1994, 3 Jan. '97, 5 Jan '98, 7 Mar 00, 5 March 01, 3 Jan 03

David Fankhauser, Professor of Biology and Chemistry

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.

EQUIPMENT: SUPPLIES:

spectrophotometer

two cuvettes, "B" and "S"

18 13 x 100 mm test tubes

25 mL graduated cylinder

37C hot block

1 mL micropipet & tips

1 mL glass pipets

stopwatch

toluenized cells on ice:

T-0, T-20, T-40, T-60

(Minutes grown in lactose)

2% K₂CO₃

20 mM orthonitrophenyl galactoside

0.1 M PO₄ buffer, pH 6.5

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

R-20 T-20 _____ 1.0 2.0 1.0 S

R-40 T-40 _____ 1.5 2.0 0.5 S

R-60 T-60 _____ 1.5 2.0 0.5 S

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.

e: When the stopwatch reads 15 minutes, withdraw 1.0 mL from R-0, add to F-0 (with K₂CO₃), mix. At 30 second intervals, repeat aliquoting for R-20, R-40 and R-60

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).

5. Calculate specific activities for each sample:

(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.