20.109(S08):Module 2
Module 1
Instructors: Alan Jasanoff and Agi Stachowiak
TA: [[]]
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 KD 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, referring to Nagai paper and sequence.
Then read about CaM-M13 and use protein viewer (also Zhang paper re: binding sites) to choose targets.
Finally, they plan primers for SDM. (or on a second day, too much at once?)
Module 2 Day 2: Site-directed mutagenesis
Students set up SDM. While it runs, have journal article discussion, and/or get calcium titration curve for WT protein using Nanodrop.
(Staff will do initial transformation into XL1-Blue and miniprep. Not sure yet if students will then make competent and transform DE3 (requires another module day), or if it will all be done by staff. On this or next day students will prepare minipreps for sequencing.)
Module 2 Day 3: Induce protein expression
(IPTG induction - meanwhile, discuss journal article and/or get WT titration curve if don't have it yet; after 2.5 h, collect and pellet samples to observe colour; run induction O/N if necessary for particular mutants)
Module 2 Day 4: Characterize protein expression
Extract protein and perform SDS-PAGE +Coomassie; purify protein (using Ni-agarose beads) and quantify amount.
Note: week off between day 4 and day 5 of lab.
Module 2 Day 5: Protein binding assay
Students arrive in pairs to set up mutant and wild-type samples, get calcium titration curves using plate reader.
Analysis and interpretation day: titration curves as well as sequencing.
MATLAB or other modeling day? Other reading or work with calcium indicators?
Alternatively, 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?)
