ASSAY OF GALACTOSIDASE IN BACTERIAL CULTURES

©David B. Fankhauser, Ph.D.
Professor of Biology and Chemistry
University of Cincinnati Clermont College,
Batavia OH 45103
 Experimental Plan for
preparation of the cells


This page has been accessed Counter times since 6 Mar 2003. 
4 March 1997, 6 Jan '98, 29 Feb 00, 3 March 01, 6 Mar 03
 Pelleted Cell ready
for resuspension

This is a preliminary experiment to determine repressed and derepressed levels of galactosidase in E coli B and optimum assay conditions for these toluenized cells.
 

EQUIPMENT:
spectrophotometer, warmed up
two cuvettes, "B" and "S" per spectrophotometer
2 16 x 150 mm test tubes in a rack
11 13x100 mm test tubes in a rack
37C hot block, warmed up
micropipets & tips (200, 1000 and 5000 uL)
repeater pipet with 10 mL tips (for 0.4 mL buffer distribution)
stopwatch
vortex
clinical centrifuge, balance		  SUPPLIES:
5 mL glucose-grown cells (CSH A1 + 0.1% glu)
     (for the class: inoculate125 mL media, aerate by shaking)
5 mL lactose-grown cells (CSH A1 + 1.0% lac)
    (for the class: inoculate125 mL media, aerate by shaking)
2% K2CO3  in a small repipet
20 mM orthonitrophenyl D-galactoside
     (1 mL per team of two in 13x100 mm test tubes)
0.1 M PO4 buffer, pH 6.5
Toluene in ground glass stoppered bottle
sterile dH2O in 10 mL repipetter

1. Prepare a table in your lab book with the additions you will make to each reaction tube:
 
Tube dH2O buffer  culture
growth cond
diluted
A660
 volume
of cells		 20 mM
ONPG		 start A415 finish A415 change A415
1 0.4 0.4 glucose
3.00 0.2


2 3.3 0.4 lactose
0.10 0.2


3 2.9 0.4 lactose
0.50 0.2



label the tubes
 2. Each team of two persons label eleven 13x100 tubes:
        a) 2 culture dilution tubes: Glu and Lac,
        b) 3 enzyme Rxn tubes: 1R, 2R & 3R,
        c) 6 K2CO3 tubes: 1S, 1F, 2S, 2F, 3S, 3F (S for Start and F for Finish samples for each reaction tube)
aliqout out the K2CO3
 3.  Add to each of the 6 K2CO3 tubes: 2 mL 2% K2CO3. [use repeater pipet]


Plan for how to prepare the cells plus illustrations of ONPG, toluene, and derepression of the lac operon.
 
 
 

Withdraw 5 mL aliquots of the overnight cultures from each of the glucose and the lactose grown cells.
Place culture aliquots in labeled16 x 150 mm tubes
Spin at low speed for 10 minutes (a setting of 4 on the clinical table top centrifuge.  (Yes, 16 x 150 mm tubes can take this low speed centrifugation, but should be used with caution.)
Discard the supernatant by decantation, save the pellet.
Add 10 mL sterile d H2O to the pellet.
Vortex thoroughly to resuspend the pellet.
Determine and record the A660 of the resuspended bacteria.

Perform dilution to make 5 mL of cell suspension, A660= 0.2. Read A660 again, record in the table.
Dilution refresher: 
      Desired dilution factor = given/desired = actual A660/0.200.
      To make 5 mL, the aliquot will equal 5 mL/dilution factor.
      To this aliquot, add sufficient diluent to make up 5 mL.
add the toluene
 Toluenize the cells: Add 3 drops of toluene to the diluted cells, cover with parafilm, cover securely, shake well and place on ice.
cover with parafilm

Set up enzyme reaction tubes: Add ingredients to the enzyme Rxn tubes as in the table you recorded in step 1, in this order: dH2O, buffer, cells. Do not add ONPG yet.

7. Prewarm the three prepared enzyme Rxn tubes in 37 C hot block for two minutes.
8. Start the reaction by adding ONPG to each tube, vortex, return to 37 C hot block.
9. Withdraw the starting blanks (Do in sequence:, you may use the same1 mL pipet for 1S, 2S & 3S.) Draw 1.00 mL from tube 1R, add to tube 1S (which contains 2 mL K2CO3), start stopwatch.
Return tube 1 to 37C hot block.
At 30 second intervals, remove 1 mL from tubes 2 and then 3, adding to tubes 2S and 3S, each with 2 mL K2CO3. Flick or vortex tubes to mix.
10. Incubate for 15 minute.
11. Withdraw the finish samples: using a fresh tip and in sequence, take 1.00 mL from tube 1, add to tube 1F. At 30 second intervals, add 1 mL aliquots from tubes 2 and 3 to tubes 2F and 3F. Flick tubes to mix.
12. Read the A415 of the six tubes (1S, 1F, 2S, 2F, 3S, 3F).
Enzyme activity is shown by the yellow. 13. Determine the change in A415 for each of the three enzyme reaction tubes.
14. Calculate specific activities for each sample:

(1/mL of cells tested) x (A415 from assay)/(A660 of the diluted culture)

 

1REAGENTS FOR LACTASE ENZYME ASSAY:

0.1 M PO4 pH 6.5 buffer:
        0.5 g KH2PO4 + 0.9 g Na2HPO4, dissolve in 100 mL H2O
20 mM o-nitrophenyl--D galactoside (ONPG):
        181 mg ONPG, dissolve 30 mL dH2O, warm slightly & swirl to dissolve.
2% K2CO3:
        dissolve 5 g K2CO3 in 250 mL dH2O, stir to dissolve.

10x Cold Spring Harbor A Medium
        (NH4)2SO4 10 g
        K 2HPO4 105 g
        KH2PO4 45 g
        MgSO4•H2O 1g
    dissolve in dH2O, q.s. to 1 L