20.109(S08):Module 2

In this experiment, you will modify a protein called inverse pericam in order to change its fluorescence properties. Inverse pericam (IPC) comprises a permuted fluorescent protein linked to a calcium sensor (cite Nagai). The “inverse” in the name refers to the fact that this protein shines brightly in the absence of calcium, but dimly once calcium is added. The dissociation constant K_D of wild-type IPC with respect to calcium is 0.2 μM (see figure below). Your goal will be to shift this titration curve by altering the calcium binding affinity of IPC’s calcium sensor portion. You will modify inverse pericam at the gene level using a process called site-directed mutagenesis, express the resultant protein in a bacterial host, and finally purify your mutant protein and assay its calcium-binding activity via fluorescence. In the course of this module, we will consider the benefits and drawbacks of different approaches to protein design, and the types of scientific investigations and applications enabled by fluorescently tagged biological molecules.

Module 2 Day 1: Start-up protein engineering

Students dissect pericam sequence into constituent parts.

Then read about CaM-M13 and use protein viewer to choose targets.

Finally, they plan primers for SDM. (or on a second day?)

Also get calcium titration curve for WT plasmid using Nanodrop --> no, do on another day (2 or 3).

Module 2 Day 2: Site-directed mutagenesis

(Staff or students do transformation/miniprep/transformation work? Timing issues, may take a second lab day...)

Module 2 Day 3: Induce protein expression

(IPTG induction - meanwhile, discuss journal article; collect and pellet samples)

Module 2 Day 4: Characterize protein expression

Extract protein and perform SDS-PAGE; purify protein and quantify amount.

Note: week off between day 4 and day 5 of lab.

Module 2 Day 5: Protein binding assay and Sequence analysis

Calcium titration curve, using plate reader. Then come back and start data analysis.

Also sequence analysis.

Module 2 Day 6: Name

MATLAB modeling day? Other reading or work with calcium indicators?

Module 2 Day 7: Name

Options as Day 6. Could have two days of intro/design, but this means pellets are sitting around for a week before protein extraction (better or worse than protein sitting around for a week?)

Module 2 Day 8: Student presentations