ING: Day 1
Materials and Methods
Students will work in pairs during the ING experiment. During the first lab period, students will set up bacterial growth cultures, perform culture additions, and collect samples for analyses.
Organization is critical on the first day: the cultures must be inoculated and transferred into the shaker within the first 30 minutes of the lab period in order to allow sufficient time to obtain a full cell growth and enzyme induction.
Organize the following materials:
a. “Assay” test tubes (18x150 mm) to collect samples for measurement of enzymatic activity from the control, antibiotics, IPTG, and lactose cultures (see Table 1). Time points will be taken at 0, 20, 50, 70, 90, and 110 min. Label a total of 24 test tubes to hold these samples, and add one drop of 1% sodium deoxycholate to each tube.
b. “Gel Analysis” test tubes (13x100 mm) for preparation of samples for gel electrophoresis. Label a total of 16 test tubes as described in Procedure Day 1.
c. “Culture Growth” test tubes (13x100 mm) for the cell growth sampling. Label a total of 24 test tubes as above.
d. Reagent tubes containing chloramphenicol, IPTG, and α-lactose.
e. An ice bucket half full of crushed ice.
Table I. Composition of Experimental Cultures
| Culture |
Medium
+ cells
(mL)(a) |
Water
(mL)(b) |
Chloramphenicol
(mL) |
IPTG
(mL) |
α-lactose
(mL) |
Start
time(c) |
| Control |
30 |
|
– |
– |
– |
|
| Antibiotic |
30 |
|
0.3 |
1.0 |
– |
|
| IPTG |
30 |
|
– |
1.0 |
– |
|
| Lactose |
30 |
|
– |
– |
1.3 |
|
(a) Your instructor will prepare a bacterial culture grown to “mid-log” phase before the class begins.
Starting A410=0.1-0.3
(b) Add water so that the total volume of added chemical and deionized water is 2 mL.
(c) Record the time the flask was introduced into the incubator bath (be sure to stagger the times by ~5min!).
Procedure: Day 1
1. Preparation of experimental cultures. You will be given ~150 mL of a cell culture in mid-log phase. IMPORTANT: Be sure to swirl the flask well to resuspend the cells before dividing up for the experimental cultures. Prepare the four cell cultures as described in Table I in 125 mL Erlenmeyer flasks. Immediately after completing the additions, take samples from each culture for the zero time cell growth (1.0 mL) and β-galactosidase activity (1.0 mL) measurements, and gel electrophoresis (3.0 mL) as described in Sections 2 through 4 below. For convenience in sampling from the cultures throughout the experiment, stagger the time you place the three culture flasks into the designated 37°C baths by about 5 min. Be sure to record the time of placement of each flask in the bath so that samples can be taken at accurately timed intervals as described in Sections 2, 3, and 4 below. NOTE: Zero time is the time at which you place the culture into the water bath, not the time at which you add the reagents.
2. Growth curve samples. Remove 1.0 mL samples from each of the four Erlenmeyer flask cultures at times of 0, 20, 50, 70, 90, and 110 minutes and place in a labeled 13x100 mm test tube (which you should have previously prepared). Keep the test tube ON ICE until an OD410 measurement can be made in order to prevent further cell growth. Made the OD410 measurements in the first lab period. Re-suspend the cells if necessary. For a sample having an absorbance greater than 0.6, dilute the sample appropriately (with water) so that the absorbance is in the range 0.1 to 0.3. Record any required dilution factors and absorbance values in DATA SHEET I. The culture fluid can be discarded into disinfectant.
3. Sampling procedure for β-galactosidase assay. Remove 1.0 mL samples from each of the four Erlenmeyer flask cultures at times of 0, 20, 50, 70, 90, and 110 minutes. Place each sample into an 18x150 mm test tube containing one drop of 1% sodium deoxycholate. Shake each tube for 30 minutes at 37°C in the designated water bath. Add one drop of toluene and shake the tube vigorously to ensure complete mixing. This treatment kills the cells and releases this particular enzyme in an active form. When all of the samples have been prepared and incubated appropriately, the batch of test tubes can be frozen and stored until the next period when β-galactosidase can be assayed as described in Procedure Day 2, below. Your instructor will tell you where to store the tubes.
4. Sampling procedure for gel electrophoresis. Remove 3.0 mL samples from each of the four Erlenmeyer flask cultures at times of 0, 50, 90, and 110 minutes. Place each sample into a labeled 13x100 mm test tube and store on ice. As is convenient, transfer approximately half of the sample into a similarly labeled 1.5 mL microcentrifuge tube and pellet the cells. Spin approximately 2 minutes. Discard the supernatant and transfer the remainder of the sample into the same microcentrifuge tube and pellet the cells. Discard the supernatant and try to dry the inside of the tube as much as possible without disturbing the cell pellet. Add 20 μL of denaturing sample buffer (containing tracking dye, SDS, and β-mercaptoethanol) and resuspend the cells using a vortexer. These denatured samples can be stored on the bench. When all of the experimental samples have been prepared, the collection of microfuge tubes can be frozen and stored until the next period when the electrophoresis gel will be performed as described in Procedure Day 2, below. Your instructor will tell you where to store the tubes.
Growth Curve Data Sheet
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