20.109(S08):Module 2
Module 2
Instructors: Alan Jasanoff and Agi Stachowiak
TA: Victor Lelyveld
In this experiment, you will modify a protein called inverse pericam (developed by Nagai et al.) in order to change its fluorescence properties. Inverse pericam (IPC) comprises a permuted fluorescent protein linked to a calcium sensor. 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.
We gratefully acknowledge 20.109 instructor Natalie Kuldell for helpful discussions during the development of this module, as well as for her work in developing a related module last year.
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.
Module 2 Day 2: Site-directed mutagenesis
Students set up SDM. While it runs, have journal article discussion.
(Staff will do initial transformation into XL1-Blue, pick colonies, and miniprep.)
Module 2 Day 3: Prepare expression system
Students prepare competent DE3 and transform with mutant IPCs.
They prepare samples for sequencing from miniprep.
They test wild-type protein fluorescence uing the Nanodrop.
Module 2 Day 4: Induce protein expression
Prior to starting induction, students do sequencing analysis to choose which mutant to pursue?
IPTG induction -after 2.5 h, collect and pellet samples to observe colour; run induction O/N if necessary for particular mutants
Meanwhile, do some kind of modeling exercise (e.g., scaled down version of rational design by energy).
Note: week off between day 4 and day 5 of lab.
Module 2 Day 5: Characterize protein expression
Extract protein and perform SDS-PAGE +Coomassie; purify protein (using Ni-agarose beads) and quantify amount.
Module 2 Day 6: Assay protein behavior
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.
