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GENE REGULATION:
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aliquot of cells growing in lactose |
This page has been accessed 31 Dec 1993, rvsd 1 Jan 1996, 3 Jan. '97, 3 Jan '98, 7 Mar 00, 6 Mar 01 |
dissolves cell membrane permitting enzyme assay |
| EQUIPMENT (per team of two): |
SUPPLIES: |
| 15 13 x 100 mm
test tubes in TT rack 37°C hot block 5 mL displacement pipetes & tips two 1 mL micropipets & tips/table stopwatch spectrophotometer two cuvettes, "B" and "S" in TT rack |
toluenized cells on ice: T-0, T-20, T-40, T-60, T-80 (Minutes grown in lactose) 2% K2CO3 20 mM orthonitrophenyl galactoside 0.1 M PO4 buffer, pH 6.0 dH2O (25 mL in 125 mL flask) |
| Per team of two, assaying B galactosidase: | |
| Four reaction tubes: |
eight K2CO3
tubes: |
| R-0 , R-20, R-40, R-60 | S0, S20, S40, S60, F0, F20, F40, F60 (start and finish) |
ASSAY OF GALACTOSIDASE IN INDUCED BACTERIAL 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.
| 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
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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 | buffer | toluenized
cells |
A415 | F - S | A415/A660/aliquot | |
| R-0 | T-0 |
|
0.4 | 3.2 | S | ||||
| F | |||||||||
| R-20 | T-20 | -- | 0.4 | 3.2 | S | ||||
| F | |||||||||
| R-40 | T-40 | 2.2 | 0.4 | 1.0 | S | ||||
| F | |||||||||
| R-60 | T-60 | 2.7 | 0.4 | 0.5 | S | ||||
| F | |||||||||
| R-80 |
T-80 |
2.7 |
0.4 |
0.5 |
S |
||||
| F |
Set up tubes: add water, then 0.4 mL buffer 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.
(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.
