20.109(S13):Module 2 - Revision history

Mark Mimee: /* Module 2 */

Mark Mimee — Tue, 05 Feb 2013 02:08:31 GMT

Module 2

←Older revision		Revision as of 02:08, 5 February 2013
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	'''Instructors:''' [[User:Shannon K. Alford \|Shannon Hughes]], [http://web.mit.edu/be/people/jasanoff.htm Alan Jasanoff], and [[User:AgiStachowiak\| Agi Stachowiak]]		'''Instructors:''' [[User:Shannon K. Alford \|Shannon Hughes]], [http://web.mit.edu/be/people/jasanoff.htm Alan Jasanoff], and [[User:AgiStachowiak\| Agi Stachowiak]]

-	'''TA:'''	+	'''TA:''' [[User:Mark_Mimee \|Mark Mimee]]

	In this experiment, you will modify a protein called inverse pericam (developed by [http://www.ncbi.nlm.nih.gov/pubmed/11248055 Nagai et al.]) in order to affect its function. 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 at sufficient concentration. 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 alter the binding curve by mutating a single residue in IPC. 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. You may find that you shift the titration curve, which corresponds to altering <math>K_D</math>; or you might change its steepness, which corresponds to changing cooperativity (and thus concentration dynamic range); finally, you might affect the maximum and/or minimum fluorescence values, thus changing the sensor's signal:noise profile (fluorescence dynamic range). You might even obliterate the response to calcium entirely! 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/pubmed/11248055 Nagai et al.]) in order to affect its function. 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 at sufficient concentration. 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 alter the binding curve by mutating a single residue in IPC. 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. You may find that you shift the titration curve, which corresponds to altering <math>K_D</math>; or you might change its steepness, which corresponds to changing cooperativity (and thus concentration dynamic range); finally, you might affect the maximum and/or minimum fluorescence values, thus changing the sensor's signal:noise profile (fluorescence dynamic range). You might even obliterate the response to calcium entirely! 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.

AgiStachowiak: /* Module 2 */

AgiStachowiak — Tue, 29 Jan 2013 00:36:00 GMT

Module 2

←Older revision		Revision as of 00:36, 29 January 2013
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	'''TA:'''		'''TA:'''
-
-	<font color=red> S13 notes: This experiment will be largely unchanged from the [[20.109%28S12%29:Module_2 \| S12 version]], with three potential exceptions: (1) The 8th day will be added back, dedicated solely to in-class analysis; (2) More than three reference mutation options will be offered, after additional pilot experiments are run; (3) Possibly the final assay will be done in Tris or similar buffer rather than water, IFF the wild-type and other reference cases are reproducible in this format. I also would like to re-frame some of the lecture and lab content to emphasize signal measurement theory. </font color>

	In this experiment, you will modify a protein called inverse pericam (developed by [http://www.ncbi.nlm.nih.gov/pubmed/11248055 Nagai et al.]) in order to affect its function. 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 at sufficient concentration. 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 alter the binding curve by mutating a single residue in IPC. 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. You may find that you shift the titration curve, which corresponds to altering <math>K_D</math>; or you might change its steepness, which corresponds to changing cooperativity (and thus concentration dynamic range); finally, you might affect the maximum and/or minimum fluorescence values, thus changing the sensor's signal:noise profile (fluorescence dynamic range). You might even obliterate the response to calcium entirely! 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/pubmed/11248055 Nagai et al.]) in order to affect its function. 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 at sufficient concentration. 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 alter the binding curve by mutating a single residue in IPC. 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. You may find that you shift the titration curve, which corresponds to altering <math>K_D</math>; or you might change its steepness, which corresponds to changing cooperativity (and thus concentration dynamic range); finally, you might affect the maximum and/or minimum fluorescence values, thus changing the sensor's signal:noise profile (fluorescence dynamic range). You might even obliterate the response to calcium entirely! 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.

Shannon K. Alford at 19:29, 24 January 2013

Shannon K. Alford — Thu, 24 Jan 2013 19:29:57 GMT

←Older revision		Revision as of 19:29, 24 January 2013
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	==Module 2==		==Module 2==

-	'''Instructors:''' [[User:Shannon K. Alford \|Shannon Hughes~~-Alford~~]], [http://web.mit.edu/be/people/jasanoff.htm Alan Jasanoff], and [[User:AgiStachowiak\| Agi Stachowiak]]	+	'''Instructors:''' [[User:Shannon K. Alford \|Shannon Hughes]], [http://web.mit.edu/be/people/jasanoff.htm Alan Jasanoff], and [[User:AgiStachowiak\| Agi Stachowiak]]

	'''TA:'''		'''TA:'''

AgiStachowiak at 18:25, 4 December 2012

AgiStachowiak — Tue, 04 Dec 2012 18:25:24 GMT

←Older revision		Revision as of 18:25, 4 December 2012
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	[[Image:20109_S12M2_titration-fit.jpg‎\|thumb\|center\|480px\|'''Fitted titration curve for IPC.''' A more sophisticated analysis using curve-fitting indeed reveals <math>K_D</math> to be ~ 0.1 μM, close to the reported value for inverse pericam.]]		[[Image:20109_S12M2_titration-fit.jpg‎\|thumb\|center\|480px\|'''Fitted titration curve for IPC.''' A more sophisticated analysis using curve-fitting indeed reveals <math>K_D</math> to be ~ 0.1 μM, close to the reported value for inverse pericam.]]

-	[[20.109(S13):~~Start-up protein engineering~~ (Day1)\| Module 2 Day 1: ~~Start-up protein engineering~~]]<br>	+	[[20.109(S13):Design mutant (Day1)\| Module 2 Day 1: Design mutant]]<br>
	[[20.109(S13):Site-directed mutagenesis (Day2)\| Module 2 Day 2: Site-directed mutagenesis]]<br>		[[20.109(S13):Site-directed mutagenesis (Day2)\| Module 2 Day 2: Site-directed mutagenesis]]<br>
	[[20.109(S13):Bacterial amplification of DNA (Day3)\| Module 2 Day 3: Bacterial amplification of DNA]]<br>		[[20.109(S13):Bacterial amplification of DNA (Day3)\| Module 2 Day 3: Bacterial amplification of DNA]]<br>

AgiStachowiak: /* Module 2 */

AgiStachowiak — Tue, 04 Dec 2012 18:14:51 GMT

Module 2

←Older revision		Revision as of 18:14, 4 December 2012
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	'''TA:'''		'''TA:'''

-	<font color=red> S13 notes: This experiment will be largely unchanged from the [[20.109%28S12%29:Module_2 \| S12 version]], with three potential exceptions: (1) The 8th day will be added back, dedicated solely to in-class analysis; (2) More than three reference mutation options will be offered, after additional pilot experiments are run; (3) Possibly the final assay will be done in Tris or similar buffer rather than water, IFF the wild-type and other reference cases are reproducible in this format </font color>	+	<font color=red> S13 notes: This experiment will be largely unchanged from the [[20.109%28S12%29:Module_2 \| S12 version]], with three potential exceptions: (1) The 8th day will be added back, dedicated solely to in-class analysis; (2) More than three reference mutation options will be offered, after additional pilot experiments are run; (3) Possibly the final assay will be done in Tris or similar buffer rather than water, IFF the wild-type and other reference cases are reproducible in this format. I also would like to re-frame some of the lecture and lab content to emphasize signal measurement theory. </font color>

	In this experiment, you will modify a protein called inverse pericam (developed by [http://www.ncbi.nlm.nih.gov/pubmed/11248055 Nagai et al.]) in order to affect its function. 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 at sufficient concentration. 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 alter the binding curve by mutating a single residue in IPC. 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. You may find that you shift the titration curve, which corresponds to altering <math>K_D</math>; or you might change its steepness, which corresponds to changing cooperativity (and thus concentration dynamic range); finally, you might affect the maximum and/or minimum fluorescence values, thus changing the sensor's signal:noise profile (fluorescence dynamic range). You might even obliterate the response to calcium entirely! 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/pubmed/11248055 Nagai et al.]) in order to affect its function. 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 at sufficient concentration. 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 alter the binding curve by mutating a single residue in IPC. 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. You may find that you shift the titration curve, which corresponds to altering <math>K_D</math>; or you might change its steepness, which corresponds to changing cooperativity (and thus concentration dynamic range); finally, you might affect the maximum and/or minimum fluorescence values, thus changing the sensor's signal:noise profile (fluorescence dynamic range). You might even obliterate the response to calcium entirely! 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.

AgiStachowiak: New page: {{Template:20.109(S13)}}
==Module 2== '''Instructors:''' Shannon Hughes-Alford, [http:...

AgiStachowiak — Tue, 04 Dec 2012 17:35:18 GMT

New page: {{Template:20.109(S13)}} <div style="padding: 10px; width: 640px; border: 5px solid #99FF66;"> ==Module 2== '''Instructors:''' Shannon Hughes-Alford, [http:...

New page

{{Template:20.109(S13)}}

<div style="padding: 10px; width: 640px; border: 5px solid #99FF66;">

==Module 2==

'''Instructors:''' [[User:Shannon K. Alford |Shannon Hughes-Alford]], [http://web.mit.edu/be/people/jasanoff.htm Alan Jasanoff], and [[User:AgiStachowiak| Agi Stachowiak]]

'''TA:'''

 S13 notes: This experiment will be largely unchanged from the [[20.109%28S12%29:Module_2 | S12 version]], with three potential exceptions: (1) The 8th day will be added back, dedicated solely to in-class analysis; (2) More than three reference mutation options will be offered, after additional pilot experiments are run; (3) Possibly the final assay will be done in Tris or similar buffer rather than water, IFF the wild-type and other reference cases are reproducible in this format 

In this experiment, you will modify a protein called inverse pericam (developed by [http://www.ncbi.nlm.nih.gov/pubmed/11248055 Nagai et al.]) in order to affect its function. 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 at sufficient concentration. 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 alter the binding curve by mutating a single residue in IPC. 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. You may find that you shift the titration curve, which corresponds to altering <math>K_D</math>; or you might change its steepness, which corresponds to changing cooperativity (and thus concentration dynamic range); finally, you might affect the maximum and/or minimum fluorescence values, thus changing the sensor's signal:noise profile (fluorescence dynamic range). You might even obliterate the response to calcium entirely! 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 prior work in developing a [http://openwetware.org/wiki/20.109:Module_2 related module].

[[Image:20109_S12M2_titration-ex.png|thumb|center|480px|'''Raw 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 μM here.]]

[[Image:20109_S12M2_titration-fit.jpg‎|thumb|center|480px|'''Fitted titration curve for IPC.''' A more sophisticated analysis using curve-fitting indeed reveals <math>K_D</math> to be ~ 0.1 μM, close to the reported value for inverse pericam.]]

[[20.109(S13):Start-up protein engineering (Day1)| Module 2 Day 1: Start-up protein engineering]] 
[[20.109(S13):Site-directed mutagenesis (Day2)| Module 2 Day 2: Site-directed mutagenesis]] 
[[20.109(S13):Bacterial amplification of DNA (Day3)| Module 2 Day 3: Bacterial amplification of DNA]] 
[[20.109(S13):Prepare expression system (Day4)| Module 2 Day 4: Prepare expression system]] 

Note: spring break week occurs between day 4 and day 5 of lab.

[[20.109(S13):Induce protein and evaluate DNA (Day5)| Module 2 Day 5: Induce protein and evaluate DNA]] 
[[20.109(S13):Purify protein (Day6)| Module 2 Day 6: Purify protein]] 
[[20.109(S13):Characterize protein expression (Day7)| Module 2 Day 7: Characterize protein expression]] 
[[20.109(S13):Assess protein function (Day8)| Module 2 Day 8: Assess protein function]] 

[[20.109(S13): Protein engineering report | Protein engineering research article]]

[[20.109(S13): TA notes for module 2| TA notes, mod 2]]

20.109(S13):Module 2 - Revision history

Mark Mimee: /* Module 2 */

AgiStachowiak: /* Module 2 */

Shannon K. Alford at 19:29, 24 January 2013

AgiStachowiak at 18:25, 4 December 2012

AgiStachowiak: /* Module 2 */

AgiStachowiak: New page: {{Template:20.109(S13)}} ==Module 2== '''Instructors:''' Shannon Hughes-Alford, [http:...

AgiStachowiak: New page: {{Template:20.109(S13)}}
==Module 2== '''Instructors:''' Shannon Hughes-Alford, [http:...