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Last time you navigated a great deal of information in order to design mutagenized inverse pericams – nice work! Today you will put your designs into practice.
Michael Smith, 1993 Chemistry Nobel Prize co-winner (with Kary Mullis, inventor of PCR) for developing site-directed mutagenesis. (© The Nobel Foundation. All rights reserved. This content is excluded from our Creative Commons license. For more information, see http://ocw.mit.edu/fairuse.)
The site-directed mutagenesis (SDM) strategy you will use shares some features with the polymerase chain reaction (PCR) for DNA amplification. Recall from Module 1 that PCR amplification involves multiple cycles of melting, annealing, and extending. To create one or more base-pair mutations in the product DNA, primers that have a slight mismatch to the original template can be used. At a low enough annealing temperature (~25 °C below the primer melting temperature), these nearly-complementary primers will still anneal to the template DNA, but the copies created during the extension phase will contain the mutation.
Today you will begin by combining plasmid DNA encoding wild-type inverse pericam with the mutagenic primers you designed. These will be acted upon by a DNA polymerase to generate mutant plasmid. Even more copies of the mutant plasmid can be made by introducing it into bacteria in a process called transformation, which we'll discuss (and do!) next time. Remember that there is still parental - that is, non-mutant - DNA present in your SDM reaction mixture. In order to propagate only the mutant plasmid upon introduction into bacteria, the parental DNA is specifically digested using the DpnI enzyme prior to bacterial transformation. (Because DpnI only digests methylated DNA, the synthetically made and thus non-methylated mutant DNA is not digested.) The resulting small linear pieces of parental DNA are simply degraded by the bacteria, whereas the largely intact (but nicked) mutant DNA is actually repaired by these very same bacteria.
Overview of the QuikChange® site-directed mutagenesis method. (Courtesy of Agilent Technologies, Inc. Used with permission.)
The thermocycling reaction today will run for a little over two hours. During this incubation time, we will discuss two articles from the primary literature. We will 'warm-up' by discussing the paper by Heim, Prasher, and Tsien (1994). This is a short paper describing the very first attempt to mutagenize GFP, and a fine introduction to some of the concepts and methods used in this module. Next we will do a close reading of the paper that introduced inverse pericam, by Nagai, et al (2001). We will examine the construction and analysis of the inverse pericam (IPC) multi-component calcium sensor in some depth.
Now might be a good time to mention why we care about measuring intracellular calcium in the first place. Calcium is involved in many signal transduction cascades, which regulate everything from immune cell activation to muscle contraction, from adhesion to apoptosis - see for example the reviews by David Clapham in Cell (2007), or Ernesto Carafoli in PNAS (2002). Intracellular calcium (Ca2+) is normally maintained at ~100 nM, orders of magnitude less than the ~mM concentration outside the cell. ATPase pumps act to keep the basal concentration of cytoplasmic calcium low. Often calcium acts as a secondary messenger, i.e., it relays a message from the cell surface to its cytoplasm. For example, a particular ligand may bind a cell surface receptor, causing a flood of calcium ions to be released from the intracellular compartments in which they are usually sequestered. These free ions in turn may promote phosphorylation or other downstream signaling.
The proteins that bind calcium do so with a great variety of affinities, and have roles ranging from sequestration to sensing. Some calcium responses may have long-term effects, particularly in the case of transcription factors that can bind calcium. As you learned last time, calmodulin works as a calcium sensor by undergoing a conformational change upon calcium binding. Your goal today is to prepare mutant calmodulin (in the context of inverse pericam) DNA, in order to alter the affinity of the resulting protein for calcium.
Heim, R., D. C. Prasher, and R. Y. Tsien. "Wavelength Mutations and Posttranslational Autoxidation of Green Fluorescent Protein." PNAS 91, no. 26 (December 20, 1994): 12501-4. [Full text]
Nagai, T., et al. "Circularly Permuted Green Fluorescent Proteins Engineered to Sense Ca2+." PNAS 98, no. 6 (March 6, 2001): 3197-3202. [Full text]
Clapham, D. E. "Calcium Signaling." Cell 131, no. 6 (December 14, 2007): 1047-1058.
Carafoli, E. "Calcium Signaling: A Tale For All Seasons." PNAS 99, no. 3 (February 5, 2002): 1115-22. [Full text]
We will be using the QuickChange® kit from Stratagene to perform our site-directed mutageneses. Each group will set up one reaction, for their chosen X#Z mutation. Meanwhile, the teaching faculty will set up a single positive control reaction, to ensure that all the reagents are working properly. You should work quickly but carefully, and keep your tube in a chilled container at all times. Please return shared reagents to the ice buckets on the front bench when you are done with them.
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During the production of the mutagenized DNA, we will discuss the two journal articles cited in the introduction. The purpose of this discussion will be two-fold: 1) to familiarize ourselves with the history of protein design, and 2) to continue to explore ways of talking about the scientific literature. (Probably you are all pros after the Module 1 journal clubs!)
As you read the paper by Heim, Prasher, and Tsien, consider the following questions.
When you arrive in lab today, each group will be assigned one of the following numbered topics to present to and discuss with the rest of the class. The same group will cover topics #1 and #8. You should be somewhat familiar with the whole Nagai et al paper by now, but will have some time in-class to refresh your memory and become the resident expert in one of the following areas (note that sometimes the Results/Discussion text pertaining to a particular figure may be out of numerical order - e.g., Fig. 4 is written up after Fig. 5):
Finally, you should all consider the similarities and differences between the research described in the papers above and the research that you are undertaking in this module.