20.109(S21):Module 3

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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 Caleb Perez

Overview

The process of scientific inquiry encompasses much more than the collection and interpretation of data. A key part of the process is designing experiments that address a hypothesis based on the collection of new information, new materials, or new technologies. In this, 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. During this module, you will analyze data collected by previous 109 students and use the information to design a new version of inverse pericam (IPC), which is a protein-based fluorescent sensor that responds to calcium levels.

Calcium is involved in many signal transduction cascades, which regulate everything from immune cell activation to muscle contraction, from adhesion to apoptosis. 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, in that 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. Calmodulin works as a calcium sensor by undergoing a conformational change upon calcium binding. Your goal in this module is to study previously generated mutants of calmodulin (in the context of IPC). These mutants were generated in an attempt to alter the affinity and / or cooperativity of the resulting protein for calcium. You will assess the success of these mutations in altering the activity of IPC and then propose a new mutation that improves upon the previous results.


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

Reviews for calcium signaling:

Notes for teaching faculty

Prep notes for M3