20.109(S08):Module 2: Difference between revisions

From OpenWetWare
Jump to navigationJump to search
No edit summary
 
(18 intermediate revisions by one other user not shown)
Line 7: Line 7:
'''Instructors:''' [http://web.mit.edu/be/people/jasanoff.htm Alan Jasanoff] and [[User:AgiStachowiak| Agi Stachowiak]]
'''Instructors:''' [http://web.mit.edu/be/people/jasanoff.htm Alan Jasanoff] and [[User:AgiStachowiak| Agi Stachowiak]]


'''TA:''' [[Victor Lelyveld]]
'''TA:''' [[User:Victor_S._Lelyveld|Victor Lelyveld]]


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.
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.


[[20.109(S08):Start-up protein engineering | Module 2 Day 1: Start-up protein engineering]]<br>
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.


Students dissect pericam sequence into constituent parts, referring to Nagai paper and sequence.
[[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.]]


Then read about CaM-M13 and use protein viewer (also Zhang paper re: binding sites) to choose targets.
[[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>
Finally, they plan primers for SDM.
[[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):Site-directed mutagenesis| Module 2 Day 2: Site-directed mutagenesis]]<br>
 
Students set up SDM. While it runs, have journal article discussion.
 
(Staff will do initial transformation into XL1-Blue, pick colonies, and miniprep.)
 
[[20.109(S08):Prepare expression system| Module 2 Day 3: Prepare expression system]]<br>
 
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.
 
[[20.109(S08):Induce protein expression| Module 2 Day 4: Induce protein expression]]<br>
 
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.
Note: week off between day 4 and day 5 of lab.


[[20.109(S08):Characterize protein expression| Module 2 Day 5: Characterize protein expression]]<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>
Extract protein and perform SDS-PAGE +Coomassie; purify protein (using Ni-agarose beads) and quantify amount.
[[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):Assay protein behavior| Module 2 Day 6: Assay protein behavior]]<br>
 
Students arrive in pairs to set up mutant and wild-type samples, get calcium titration curves using plate reader.
 
[[20.109(S08):Sequence and titration analysis| Module 2 Day 7: Sequence and titration analysis]]<br>
 
Analysis and interpretation day: titration curves as well as sequencing.


[[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

Home        People        Schedule Spring 2008        Assignments        Lab Basics        OWW Basics       
DNA Engineering        Protein Engineering        Biomaterials Engineering              

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

Retrieved from "https://openwetware.org/mediawiki/index.php?title=20.109(S08):Module_2&oldid=190410"