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7.13 experimental microbial genetics project lab

WARNING NOTICE: The experiments described in these materials are potentially hazardous and require a high level of safety training, special facilities and equipment, and supervision by appropriate individuals. You bear the sole responsibility, liability, and risk for the implementation of such safety procedures and measures. MIT shall have no responsibility, liability, or risk for the content or implementation GUS ASSAY
The Quasi Definitive Guide to Determining β-Glucuronidase Activity in Cell Lysates (special bonus protocols for the harvesting and lysis of E. coli, Rhodococcus and Corynebacterium) by Jennie Cho, (modified and spiffed up by xian o’brien, selflessly edited by Phil Lessard)
Adapted from Wilson et al., The Escherichia coli gus Operon: Induction and Expression of the gus Operon in E.coli and
the Occurrence and Use of GUS in Other Bacteria. From GUS Protocols: Using the GUS Gene as a Reporter of
Gene Expression
. Ed. Sean R. Gallagher, Academic Press, Inc., 1992.

Harvesting of Cultures
4. At appropriate time points during the culture, remove 1.6 ml of culture into a 2ml
5. Working quickly, briefly vortex the 1.6 ml sample and remove 100 uL into a spectrophotometer cuvette containing 900 uL fresh culture media. Spin down the remaining 1.5 ml sample, remove the supernatant and place the pellet immediately at -80°C. (Note: Samples will keep at -80°C indefinitely; do NOT freeze at -20°C.) 6. Measure and note the OD600 of the 1:10 dilution. (Note: The OD600 of your sample will factor into the later calculation of GUS activity.) Lysing of Cells
8. Thaw sample pellets on ice.
9. Resuspend pellets in 374 uL B-PER™ Bacterial Protein Extraction Reagent (Pierce product
78248), 50 uL Protease and Phosphatase Inhibitor Cocktail for use with bacterial cell extracts (Sigma product P8465; reconstituted per manufacturer’s indications), 1 uL 34 mg/mL chloramphenicol (prepared in methanol), and 6 uL 10mg/ml lysozyme (prepared fresh in dH20; can be omitted when working with E. coli). 10. Vortex vigorously for 1 min. 11. Incubate on ice for 5 min. 12. At this point, what you have is a crude lysate slurry that can be assayed for GUS activity using the procedure in the next section yielding a measure of activity in terms of nM product formed/minute · OD600 unit. (Note: For Corynebacterium and Rhodococcus this is the best you can hope for using this lysis procedure). 13. For E. coli samples, spin down the crude lysate 1 min and place on ice. 14. What you have now is a cleared lysate (the supernatant cleared of cell debris). The protein concentration of the cleared lysate can be determined using the Bradford assay (see the separate protocol for this assay). The cleared lysate can be assayed for GUS activity using the procedure described in the next section, yielding a measure of activity in terms of nM product formed/minute · mg protein. (Note: Activity per mg protein is a more desirable measure of activity than activity per OD600 unit. Think about it.) P . L E S S A R D 2 0 0 2 , A L L R I G H T S R E S E R V E D
GUS Assay
1. Keep cell lysates on ice.
2. Prepare fresh GUS assay buffer and prewarm to 37°C.
3. Prepare disposable cuvettes (at least 3 per assay), each containing 800 uL 0.4 M Na2CO3
4. Prepare one 2 mL Eppendorf tube per sample containing 800 uL of the GUS assay buffer prepared as above. Add 200 uL cell lysate to the tube. (This gives a final concentration of PNPG 1mM.) Note time. 5. Place reactions at 37°C and note the time. (Note: A water bath is preferable, but consistency is key.) Watch for development of yellow color (p-nitrophenol). At no fewer than 3 time points, (e.g. 15, 30, 45 minutes) remove 100 uL from each sample into a spectrophotometer cuvette. (Note: When assaying crude lysate slurry, tap spin the reaction tubes and remove 100 uL of the supernatant into the spectrophotometer cuvette.) Briefly vortex the reaction tubes and return to 37°C quickly. The time points do not have to be equally spaced, but be sure to note each time. 6. Measure absorbance at 405 nm for each time point against a stopped blank reaction. (Note: The color formation is stable in the stop solution. Less error is introduced if you measure the absorbance of all of the time points at once rather than after each time point.) Calculating the Rate of Reaction
1. For each sample, plot a graph of OD405 (y-axis) vs. time (x-axis). Calculate the slope S of the
graph (which should be linear!) in OD405 units per minute. (Or cheat and have Excel calculate it for you) 2. The rate of reaction R (in nanomoles product per minute per OD600 unit)** for each sample is where V is the volume assayed in milliliters. In this case, V = 0.02 mL because 0.2 mL sample is used in the initial reaction (step 3) and one-tenth of this (100 uL) is removed into stop solution for each time point (step 4). The 0.02 in the denominator has been derived from the molar extinction coefficient of p-nitrophenol. Under the conditions specified by this particular protocol, the molar extinction coefficient of p-nitrophenol is 18,000. In the 900 uL final volume, an absorbance of 0.02 represents 1 nmol of product produced. **The rate of reaction R can also be calculated in terms of nanomoles product per minute per mg protein: R = S/(0.02 x V x protein concentration) The Bradford Assay can be used to determine the protein concentration of each sample. P . L E S S A R D 2 0 0 2 , A L L R I G H T S R E S E R V E D
Reagents
GUS Assay Buffer
1M dithiothreitol (DTT)
Prepare ahead and store 1 mL aliquots at -20°C. Prepare fresh and prewarm to 37°C
100mM p-nitrophenyl β-D-glucuronide
GUS Buffer Stock Solution
Prepare fresh and keep on ice.
0.4 M Na2CO3 (Stop Solution)
Prepare ahead and store at room temperature. DTT and PNPG need to be added before use. Prepare ahead and store at room temperature. 0.5M NaH2PO4
10mg/mL lysozyme
(if working with Rhodococcus or Corynebacterium) Prepare ahead and store at room temperature. Prepare fresh and keep on ice.
0.5M Na2HPO4
B-PER™ Bacterial Protein Extraction Reagent
Pierce ready-to-use product 78148, 500mL Prepare ahead and store at room temperature. Protease and Phosphatase Inhibitor Cocktail
for use with bacterial cell extracts
0.5M ethylenediamine tetraacetic acid (EDTA)
Reconstituted per manufacturer’s indications Prepare ahead and store 200 uL aliquots at -80° Prepare ahead and store at room temperatu e 34 mg/ml chloramphenical
for 10 mL
340mg chloramphenical
methanol to 10mL
Prepare ahead and store 1 mL aliquots at -20°C.
P . L E S S A R D 2 0 0 2 , A L L R I G H T S R E S E R V E D

Source: http://mitocw.espol.edu.ec/courses/biology/7-13-experimental-microbial-genetics-fall-2003/labs/GUS_Assays_v2.pdf

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