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  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.
 

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	1.4	0.4	glucose		2.00	0.2
2	3.3	0.4	lactose		0.10	0.2
3	2.4	0.4	lactose		1.00	0.2

2. Label eleven 13x100 tubes:
        a) 2 culture dilution tubes: Glu and Lac,
        b) 3 enzyme Rxn tubes: 1, 2 & 3,
        c) 6 K₂CO₃ tubes: 1S, 1F, 2S, 2F, 3S, 3F (S for Start and F for Finish samples for each reaction tube)
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 the A660 of the resuspended bacteria.
	Perform dilution to make 3mL of cell suspension, A660= 0.2. Read A660 again, record in the table.
	Toluenize the cells: Add 3 drops of toluene to the diluted cells, cover with parafilm, shake well and place on ice.
	Set up enzyme reaction tubes: Add ingredients to the enzyme Rxn tubes as in the table in this order: dH2O, buffer, cells. Do not add ONPG yet.
	7. Prewarm the three prepared enzyme Rxn tubes in 37C hot block for two minutes. 8. Start the reaction by adding ONPG to each tube, vortex, return to 37C hot block.. 9. Withdraw the starting blanks (a single 1 mL micropipet may be used for this stage if done in sequence): take 1.00 mL from tube 1, add to tube 1S with 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 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).
	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
 
 
 

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