20.109(S08):Module 2: Difference between revisions

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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<sub>D</sub> 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==


[[20.109(S08):Start-up protein engineering | Module 2 Day 1: Start-up protein engineering]]<br>
'''Instructors:''' [http://web.mit.edu/be/people/jasanoff.htm Alan Jasanoff] and [[User:AgiStachowiak| Agi Stachowiak]]


Students dissect pericam sequence into constituent parts, referring to Nagai paper and sequence.
'''TA:''' [[User:Victor_S._Lelyveld|Victor Lelyveld]]


Then read about CaM-M13 and use protein viewer (also Zhang paper re: binding sites) to choose targets.
In this experiment, you will modify a protein called inverse pericam (developed by [http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=11248055&ordinalpos=5&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum  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 <math>K_D</math> of wild-type IPC with respect to calcium is reported to be 0.2 μM (see also 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.


Finally, they plan primers for SDM. (or on a second day, too much at once?)
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 [http://openwetware.org/wiki/20.109:Module_2 related module] last year.


[[20.109(S08):Site-directed mutagenesis| Module 2 Day 2: Site-directed mutagenesis]]<br>
[[Image:20.109 Ca-IPC-Titr-Fig.png|thumb|center|480px|'''Titration curve for IPC.''' Shown here is sample data from the teaching lab: normalized fluorescence for wild-type inverse pericam as a function of calcium concentration. As you will later learn, an apparent <math>K_D</math> can be estimated from such a plot: it is the point on the ''x''-axis where the curve crosses ''y'' = 50%, or ~0.1 &mu;M here.]]  


Students set up SDM. While it runs, have journal article discussion, and/or get calcium titration curve for WT protein using Nanodrop.
[[20.109(S08):Start-up protein engineering (Day1)| Module 2 Day 1: Start-up protein engineering]]<br>
 
[[20.109(S08):Site-directed mutagenesis (Day2)| Module 2 Day 2: Site-directed mutagenesis]]<br>
(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.)
[[20.109(S08):Prepare expression system (Day3)| Module 2 Day 3: Prepare expression system]]<br>
 
[[20.109(S08):Induce protein expression (Day4)| Module 2 Day 4: Induce protein expression]]<br>
[[20.109(S08):Characterize protein expression| Module 2 Day 3: Induce protein expression]]<br>
 
(IPTG induction - meanwhile, discuss journal article and/or get WT titration curve if don't have it yet; collect and pellet samples)
 
[[20.109(S08):Protein binding assays| Module 2 Day 4: Characterize protein expression]]<br>
 
Extract protein and perform SDS-PAGE; purify protein and quantify amount.


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


[[20.109(S08):Sequence analysis| Module 2 Day 5: Protein binding assay and Sequence analysis]]<br>
[[20.109(S08):Characterize protein expression (Day5)| Module 2 Day 5: Characterize protein expression]]<br>
 
[[20.109(S08):Assay protein behavior (Day6)| Module 2 Day 6: Assay protein behavior]]<br>
Calcium titration curves for mutants and wild-type, using plate reader.
[[20.109(S08):Data analysis (Day7)| Module 2 Day 7: Data analysis]]<br>
 
[[20.109(S08):Student presentations (M2D8)| Module 2 Day 8: Student presentations]]<br>
[[20.109(S08):Name| Module 2 Day 6: Name]]<br>
 
Analysis and interpretation day: titration curves as well as sequence analysis.
 
[[20.109(S08):Name| Module 2 Day 7: Name]]<br>
 
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?)


[[20.109(S08):Student presentations| Module 2 Day 8: Student presentations]]<br>
[[20.109(S08): TA notes for module 2| TA notes, mod 2]]

Latest revision as of 08:15, 11 March 2008


20.109(S08): Laboratory Fundamentals of Biological Engineering

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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 [math]\displaystyle{ K_D }[/math] of wild-type IPC with respect to calcium is reported to be 0.2 μM (see also 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.

Titration curve for IPC. Shown here is sample data from the teaching lab: normalized fluorescence for wild-type inverse pericam as a function of calcium concentration. As you will later learn, an apparent [math]\displaystyle{ K_D }[/math] can be estimated from such a plot: it is the point on the x-axis where the curve crosses y = 50%, or ~0.1 μM here.

Module 2 Day 1: Start-up protein engineering
Module 2 Day 2: Site-directed mutagenesis
Module 2 Day 3: Prepare expression system
Module 2 Day 4: Induce protein expression

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

Module 2 Day 5: Characterize protein expression
Module 2 Day 6: Assay protein behavior
Module 2 Day 7: Data analysis
Module 2 Day 8: Student presentations

TA notes, mod 2

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