Introduction - You have been hired by Rife Labs, Inc. to look at changes in expression of the enzyme, ribulose bisphosphate carboxylase (RUBISCO). As you may remember from your Cell and Molecular biology course, RUBISCO is the most abundant protein on the planet and makes up 40-80% of the proteins found in leaf tissue. The job of the enzyme is to capture and fix carbon in the photosynthetic pathway but the enzyme performs this function very slowly. The enzyme is non-specific for carbon dioxide and can just as easily fix oxygen instead. This oxygenase activity is a problem for plants in very warm climates where the stomata of the plant must be closed during the day increasing the amount of oxygen found inside the plant. Cam and C4 plants have found ways around this problem by fixing carbon at different times of day or in specialized places in the plant.

Rubisco is a complex protein made up of eight small subunits and eight large subunits. The small subunits are encoded by the nuclear genome and the large subunits are encoded by the chloroplast genome. You will be focusing your efforts on studying the RUBISCO large subunit (RBCL).

Your first project in Rife-Bechtel Labs, Inc. is to help to contribute to our scientific understanding of how, and when, and why RBCL expression may change in a plant. There are many example questions which have been approved for funding at the Rife-Bechtel Labs in the past, including questions: At what stage of plant growth does RBCL get expressed? Is there more or less RBCL in the leaves of a plant exposed to light compared to a plant grown in the dark? Does heat, watering, or nutrient content in the soil have an effect on RBCL expression? Do plants with different pigments in their leaves have different RBCL levels in the differently pigmented areas? Is RBCL expressed in stems, flowers?...

For your first task in your new job you will be required to work together in groups of three to design a hypothesis and come up with a proposal to test this hypothesis to answer a question about how RBCL expression changes in a plant. You will need to find two articles in the primary literature to back your hypothesis. As a part of your proposal you will need to choose and locate the plants needed for your experiment. You are allowed, with permission, to use plants in the greenhouse, or you can bring them from home, the grocery store, etc... Based on your hypothesis, you should choose between three to five different plant tissues to use to perform the following experiments.

Figure 2- Reminder of the Central Dogma of Molecular Biology and how genes are regulated.

Experimentation:

A. Examination of the Numbers of Chloroplast in your Plant Tissues of Choice (Three to Four plant tissues should be chosen per group)

You may remember that not all cells contain the same amount of any one organelle. To determine if we see any changes in RBCL expression due to transcriptional regulation, we first need to learn how many copies of the RBCL gene (ie. how many chloroplasts) are present in the cells we are interested in examining. To this end, you will harvest the DNA from your plant tissues of choice and perform Real-Time PCR in order to quantitate the number of chloroplasts present in your plant tissue.

B. Examination of the Amount of RBCL-Large Subunit Protein in your Plant Tissue of Choice (Three to five plant tissues should be chosen per group)

In order to see if the actual expression of the RBCL protein changes over time, Western Blotting Techniques will be used to quantitate how much large subunit protein is found in each tissue.

Proposal - In order to obtain funding for this project, a short description, no more than 1 page double spaced on what you plan to do for your project is due to your instructor by Friday at 5 p.m. of the first week of classes. This proposal should include background information, your hypothesis and the plant species you are going to use in your experiments. You should also include information about the types of experiments you are going to do and how these experiments will answer the question proposed. It should also include a literature cited section with citations listed to two primary literature articles.

Funding- 10 points will be awarded for this proposal.

Final report- At the end of the project, the company requests information about how your project is proceeding in the form of an E-poster lab report designed by your whole group. In this report, you will tell the world whether or not you still believe transcription of the large subunit changes in the scenerio you set out. This report will be due two weeks after we finish the experimentation. It will first be graded by your peers and by your instructors.

Other information - You have plant material in the greenhouse at your disposal and may also bring plants from home or purchase them in the grocery store. Ideally you need about 2g of tissue 6-8 oak-size leaves for all experimentation. The tissue should be kept cold after picking it from the plant and can be frozen following harvest at -80 degrees. We can get by with less tissue if you're working with certain plants, but please consult your instructor if you don't think you will have 2 g of tissue. You should choose between three to four plants or plant tissues that you want to use to isolate protein and DNA. Any more than that and it is difficult to get everything done in a single class period. If you choose to use plants in the JMU Greenhouse you must ask permission from Mark Brubaker or Heather Griscom. You should use your textbook and the internet to find journal articles or other sources that will help you support your hypothesis and learn more about Rubisco and its large subunit. Feel free to use information from past web posters to aid you in developing your proposals.