BISC 219/2009: Mod 3 Experiment 2 Assaying the transgenic plants

β-Glucuronidase (GUS) Activity Assays of Transformed Plants
The goal of the two assays for GUS activity that we will perform in the next few weeks is to obtain functional evidence that your kanamycin-resistant plants are transgenic and that your reporter gene (gusA) is being expressed by testing for the function of the protein encoded by gusA, β-glucuronidase (GUS). The gusA gene of E. coli, which encodes a β-glucuronidase (GUS), is a valuable reporter gene for use in plant systems for several reasons. β-glucuronidase activity is not detected in most plant species, therefore the detection of GUS activity in plant extracts is excellent evidence that the gusA gene has been introduced and is being expressed. Furthermore, GUS activity is a very easy to assay because a wide variety of substrates for spectrophotometric, fluorometric and histochemical assays are available. GUS specifically hydrolyzes β-conjugated D-glucuronides. We will use two GUS activity assays that use different alternative substrates. One is p-nitrophenyl glucuronide (PNPG). GUS catalyzes the cleavage of the glucuronide moiety from this substrate releasing p-nitrophenol which absorbs at 415 nm (see illustration below). We will follow the production of the yellow reaction product, p-nitrophenol, by measuring the A415 in leaf extracts. The second way we will measure gusA expression is through a histochemical semi-quantitative assay using a different substrate for beta glucuronidase, X-glucuronide (5Bromo4 chloro3indolyl-beta-D glucuronide in DMSO). This substrate, like PNPG, is colorless until cleaved by beta glucuronidase, but this time the cleavage product is blue rather than yellow. We will use a relative scale to judge amount of blue color in intact leaf tissue in this assay.

GUS Activity Assay by Histochemistry
''The goal of this procedure is to obtain additional evidence that your cloned plants are transgenic through an additional β-glucuronidase activity assay. This assay is only semi-quantitative, but it allows testing whole leaf tissue directly.'' Gus Activity Histochemical Stain: X-glucoronide (5_Bromo4 chloro3indolyl-beta-D glucoronide in DMSO), 1 M sodium phosphate (pH 7), 0.05% Triton X100. The other GUS activity assay by spectrophotometry will be performed using the following protocols Leaf Extract Preparation Spectrophotometric Assay for GUS activity Calculations
 * 1) Punch one leaf disk from each of the same plants on which you performed the other enzyme activity assay (1 control and 4 putative transformants). Place each disk into the well of a microtiter plate containing the GUS assay mixture (X-glucoronide= 5-bromo 4-chloro 3-indolyl beta-D glucoronide in DMSO) Note the well number for each sample in your notebook.
 * 2) Place the microtiter dish under vacuum to remove the air trapped within the tissue.
 * 3) Wrap the dish in saran wrap and place in the 37 C incubator for 24 hours.
 * 4) Stop the assay and extract the chlorophyll by removing the assay solution and replacing it with 75% ETOH. It will take several hours for the chlorophyll to be extracted from the tissue thus making the blue GUS enzyme reaction product readily visible.
 * 5) After a 24 reaction period score each disk for intensity of blue color using the following relative scale:  3+ very blue; 2+ blue; 1+  slightly blue; 0 not blue