Difference between revisions of "20.109(S21):Module 3"

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M3D1: [[20.109(S21):M3D1 |Review inverse pericam (IPC) cloning strategy ]] <br>
 
M3D1: [[20.109(S21):M3D1 |Review inverse pericam (IPC) cloning strategy ]] <br>
 
M3D2: [[20.109(S21):M3D2 |Examine IPC mutations ]] <br>
 
M3D2: [[20.109(S21):M3D2 |Examine IPC mutations ]] <br>
M3D3: [[20.109(S21):M3D3 |Induce and purify IPC variants ]] <br>
+
M3D3: [[20.109(S21):M3D3 |Prepare expression system and purify IPC variants ]] <br>
 
M3D4: [[20.109(S21):M3D4 |Evaluate effect of mutations on IPC variants ]] <br>
 
M3D4: [[20.109(S21):M3D4 |Evaluate effect of mutations on IPC variants ]] <br>
 
M3D5: [[20.109(S21):M3D5 |Design new IPC variant ]] <br>
 
M3D5: [[20.109(S21):M3D5 |Design new IPC variant ]] <br>

Revision as of 21:58, 2 February 2021

20.109(S21): Laboratory Fundamentals of Biological Engineering

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Spring 2021 schedule        FYI        Assignments        Homework        Communication |        Accessibility

       M1: Antibody engineering        M2: Drug discovery        M3: Protein engineering       


Module 3

Lecturer: Prof. Alan Jasanoff
Instructors: Dr. Noreen Lyell, Dr. Leslie McClain, and Dr. Becky Meyer
TAs: Jeff Hsaio and Malek Kabani

Overview

(briefly reviewed here).

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 this review by David Clapham in Cell, or this one by Ernesto Carafoli in PNAS. 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 discussed in lecture, calmodulin works as a calcium sensor by undergoing a conformational change upon calcium binding. Your goal today is to design mutation primers that will generate mutant calmodulin (in the context of inverse pericam) DNA, in an attempt to alter the affinity and/or cooperativity of the resulting protein for calcium.


The process of scientific inquiry encompasses much more than the collection and interpretation of data. A key part of the process is design – specifically of experiments that address a hypothesis and of new materials or technologies. Moreover, any design is subject to continued revision. You might redesign an experiment or tool based on your own research, or you might consult the vast body of scientific literature for other perspectives. As the old graduate student saying (sarcastically) goes, “A month in the lab might save you a day in the library!” In other words, although the process of combining the literature can be arduous or even tedious at times, it beats wasting a month of your time repeating experiments previously demonstrated not to work or reinventing the wheel.

During this module, you will generate and test a new version of inverse pericam (IPC).


Research goal: Perform site-directed mutagenesis to alter the properties of a protein-based fluorescent sensor

Image generated using BioRender.


Lab links: day by day

M3D1: Review inverse pericam (IPC) cloning strategy
M3D2: Examine IPC mutations
M3D3: Prepare expression system and purify IPC variants
M3D4: Evaluate effect of mutations on IPC variants
M3D5: Design new IPC variant

Assignments

Research proposal presentation
Mini-report

References

Notes for teaching faculty

Prep notes for M3