20.109(S08):Module 2: Difference between revisions

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(New page: Module 2 Day 1: Start-up protein engineering<br> (Initial reading about CaM-M13? Get calcium titration curve for WT plasmid? Need this day?...)
 
 
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[[20.109(S08):Start-up protein engineering | Module 2 Day 1: Start-up protein engineering]]<br>
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(Initial reading about CaM-M13? Get calcium titration curve for WT plasmid? Need this day?)
==Module 2==


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


(Protein viewer, further reading about CaM-M13 to choose targets, plans primers for SDM)
'''TA:''' [[User:Victor_S._Lelyveld|Victor Lelyveld]]


[[20.109(S08):Site-directed mutagenesis| Module 2 Day 3: Site-directed mutagenesis]]<br>
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.


(Staff or students do transformation/miniprep/transformation work? Timing issues, may take another lab day...)
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):Characterize protein expression| Module 2 Day 4: Characterize protein expression]]<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.]]  


(IPTG induction, SDS-PAGE)
[[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>
[[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):Protein binding assays| Module 2 Day 5: Protein binding assays]]<br>
Note: week off between day 4 and day 5 of lab.


(Calcium titration curve, fluorescence-based)
[[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>
[[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):Sequence analysis| Module 2 Day 6: Sequence analysis]]<br>
[[20.109(S08): TA notes for module 2| TA notes, mod 2]]
 
(Also lipofect wild-type plasmid to mammalian cells?)
 
[[20.109(S08):Name| Module 2 Day 7: Name]]<br>
 
(Microscopy assays with mammalian cells? MATLAB modeling?)
 
[[20.109(S08):Student presentations| Module 2 Day 8: Student presentations]]<br>

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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