User:Wesley J. Houston/Notebook/MAP - Revision history

Wesley J. Houston: /* Notes */

2013-01-29T04:22:14Z

Notes

←Older revision		Revision as of 04:22, 29 January 2013
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	==Notes==		==Notes==
	* This project is currently in progress.		* This project is currently in progress.
-	** Click on the individual dates, beginning on 1.24.13, to see ~~specifics~~ protocols used.	+	** Click on the individual dates, beginning on 1.24.13, to see specific protocols used.

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Wesley J. Houston at 04:22, 29 January 2013

2013-01-29T04:22:01Z

←Older revision		Revision as of 04:22, 29 January 2013
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	==Project Description/Abstract==		==Project Description/Abstract==
-	This Master's applied project (MAP) will focus on building a histone methylation-associated reporter gene. The purpose of this is to test chromatin protein function. These synthetic ~~chromatin~~ proteins will have been constructed to test varying attributes: amino acid sequence of the methyl-histone binding domain, binding domain copy number, relative positioning of tandem binding domains and activation domain strength. In order to test the amino acid sequence of the methyl-histone binding domain, polycomb chromodomains (PCDs) will be taken from samples of human, zebrafish and Drosophilia tissue.	+	* This Master's applied project (MAP) will focus on building a histone methylation-associated reporter gene. The purpose of this is to test chromatin protein function. These synthetic chtomatin proteins will have been constructed to test varying attributes: amino acid sequence of the methyl-histone binding domain, binding domain copy number, relative positioning of tandem binding domains and activation domain strength. In order to test the amino acid sequence of the methyl-histone binding domain, polycomb chromodomains (PCDs) will be taken from samples of human, zebrafish and Drosophilia tissue.

-	Amino acid sequences vary outside of the binding ~~region~~ in PCDs, but have conserved 3-D structures and methyl-histone binding surfaces. Tandem PCDs will vary the PCD copy number; their relative positioning will be determined by short or long rigid alpha helix, or flexible ~~(glycine-serine)~~ peptide linkers. The expression of the synthetic chromatin protein's target gene will be varied by the use of various activation modules (such as VP64, SP1 and p65).	+	* Amino acid sequences vary outside of the binding regions in PCDs, but have conserved 3-D structures and methyl-histone binding surfaces. Tandem PCDs will vary the PCD copy number; their relative positioning will be determined by short or long rigid alpha helix, or flexible peptide linkers. The expression of the synthetic chromatin protein's target gene will be varied by the use of various activation modules (such as VP64, SP1 and p65).
-		+
-	The activity of these synthetic chromatin proteins will be determined by the reporter gene that is to be built during the course of this project. Each protein will be expressed in transgenic cells that carry the gene in question, which produces cyan fluorescent protein upon activation. This gene will then be placed into a chromosomal FRT site in U2OS cells by way of enzymatic Flp-mediated recombination. The reporter's promoter will create histone methylation, either by the Gal4-EED fusion protein system or the human histone methylation signal.	+
-		+
-	~~A Hill equation will be used to determine reporter output:~~	+
-		+
-	~~f(X)=βXn/(Kn+Xn)~~	+

-	In the ~~resultant plot, red fluorescent~~ protein will ~~indicate the chromatin protein concentration (X) compared against~~ the cyan ~~fluorescence from the reporter~~. ~~β indicates the maximum~~ reporter ~~output~~, ~~K is~~ the ~~activation coefficient and n is~~ the ~~Hill coefficient~~.	+	* The activity of these synthetic chromatin proteins will be determined by the reporter gene that is to be built during the course of this project. Each protein will be expressed in transgenic cells that carry the gene in question, which produces cyan fluorescent protein upon activation. This gene will then be placed into a chromosomal FRT site in U2OS cells by way of enzymatic Flp-mediated recombination. The reporter's promoter will create histone methylation, either by the Gal4-EED fusion protein system or the human histone methylation signal.

-	This project will help to understand chromatin protein engagement in living cells. The protein binding dynamics of Polycomb will also be explored, which plays a major role in cancer and tissue development. Finally, the amino acid primary structure and activation domain strength will be investigated, as well as linked domains. These design principles will be defined and quantitatively described, which will provide us with more information about natural chromatin components, making the engineering of chromatin a possibility.	+	* A Hill equation will be used to determine reporter output: <math>f(X)=βX<sup>n</sup>/(K<sup>n</sup>+X<sup>n</sup>)</math>
		+
		+	* In the resultant plot, red fluorescent protein will indicate the chromatin protein concentration (X) compared against the cyan fluorescence from the reporter. β indicates the maximum reporter output, K is the activation coefficient and n is the Hill coefficient.
		+
		+	* This project will help to understand chromatin protein engagement in living cells. The protein binding dynamics of Polycomb will also be explored, which plays a major role in cancer and tissue development. Finally, the amino acid primary structure and activation domain strength will be investigated, as well as linked domains. These design principles will be defined and quantitatively described, which will provide us with more information about natural chromatin components, making the engineering of chromatin a possibility.

	==Notes==		==Notes==

Wesley J. Houston: /* Project Description/Abstract */

2013-01-29T04:03:04Z

Project Description/Abstract

←Older revision		Revision as of 04:03, 29 January 2013
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	==Project Description/Abstract==		==Project Description/Abstract==
-	This project ~~involves creating~~ a reporter gene to ~~determine~~ chromatin protein function.	+	This Master's applied project (MAP) will focus on building a histone methylation-associated reporter gene. The purpose of this is to test chromatin protein function. These synthetic chromatin proteins will have been constructed to test varying attributes: amino acid sequence of the methyl-histone binding domain, binding domain copy number, relative positioning of tandem binding domains and activation domain strength. In order to test the amino acid sequence of the methyl-histone binding domain, polycomb chromodomains (PCDs) will be taken from samples of human, zebrafish and Drosophilia tissue.
		+
		+	Amino acid sequences vary outside of the binding region in PCDs, but have conserved 3-D structures and methyl-histone binding surfaces. Tandem PCDs will vary the PCD copy number; their relative positioning will be determined by short or long rigid alpha helix, or flexible (glycine-serine) peptide linkers. The expression of the synthetic chromatin protein's target gene will be varied by the use of various activation modules (such as VP64, SP1 and p65).
		+
		+	The activity of these synthetic chromatin proteins will be determined by the reporter gene that is to be built during the course of this project. Each protein will be expressed in transgenic cells that carry the gene in question, which produces cyan fluorescent protein upon activation. This gene will then be placed into a chromosomal FRT site in U2OS cells by way of enzymatic Flp-mediated recombination. The reporter's promoter will create histone methylation, either by the Gal4-EED fusion protein system or the human histone methylation signal.
		+
		+	A Hill equation will be used to determine reporter output:
		+
		+	f(X)=βXn/(Kn+Xn)

-	~~Simply put~~, the ~~idea is that~~ the cyan ~~fluorescent~~ protein will be ~~expressed as~~ the chromatin ~~protein attaches to~~ the ~~reporter~~.	+	In the resultant plot, red fluorescent protein will indicate the chromatin protein concentration (X) compared against the cyan fluorescence from the reporter. β indicates the maximum reporter output, K is the activation coefficient and n is the Hill coefficient.
		+
		+	This project will help to understand chromatin protein engagement in living cells. The protein binding dynamics of Polycomb will also be explored, which plays a major role in cancer and tissue development. Finally, the amino acid primary structure and activation domain strength will be investigated, as well as linked domains. These design principles will be defined and quantitatively described, which will provide us with more information about natural chromatin components, making the engineering of chromatin a possibility.

	==Notes==		==Notes==

Wesley J. Houston: /* Notes */

2013-01-29T01:53:03Z

Notes

←Older revision		Revision as of 01:53, 29 January 2013
Line 20:		Line 20:
	==Notes==		==Notes==
	* This project is currently in progress.		* This project is currently in progress.
-	** Click on the individual dates, beginning on 1.24.13 to see specifics protocols used.	+	** Click on the individual dates, beginning on 1.24.13, to see specifics protocols used.

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Wesley J. Houston: /* Notes */

2013-01-29T01:52:53Z

Notes

←Older revision		Revision as of 01:52, 29 January 2013
Line 19:		Line 19:

	==Notes==		==Notes==
-	* ~~Place some notes here for visitors~~	+	* This project is currently in progress.
-		+	** Click on the individual dates, beginning on 1.24.13 to see specifics protocols used.
-	* Example: This project is currently ~~on hold until further notice~~.	+
-		+

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Wesley J. Houston: /* Project Description/Abstract */

2013-01-29T01:51:46Z

Project Description/Abstract

←Older revision		Revision as of 01:51, 29 January 2013
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	==Project Description/Abstract==		==Project Description/Abstract==
-	* Place short description of project or notes regarding this project	+	This project involves creating a reporter gene to determine chromatin protein function.

-	* Lorem ipsum dolor sit amet, consectetuer adipiscing elit. Donec porta commodo tellus. Nam a est eget libero mollis tincidunt. Aliquam purus. Quisque nulla ligula, facilisis in, pulvinar sed, molestie a, quam. Vestibulum at pede. In in sem eget odio eleifend placerat. Phasellus ultricies felis quis sapien. Etiam molestie volutpat quam. Praesent pulvinar scelerisque mi. Nam mi urna, fringilla eu, mattis sed, venenatis id, nunc. Maecenas velit eros, congue ut, placerat in, ornare vel, sem. Aenean porta enim sit amet felis gravida posuere. Phasellus faucibus nibh et orci.	+	Simply put, the idea is that the cyan fluorescent protein will be expressed as the chromatin protein attaches to the reporter.

	==Notes==		==Notes==

Wesley J. Houston: Autocreated Lab Notebook name=User:Wesley J. Houston/Notebook/MAP, content from MediaWiki:ProjectContentDefault

2013-01-24T04:43:17Z

Autocreated Lab Notebook name=User:Wesley J. Houston/Notebook/MAP, content from MediaWiki:ProjectContentDefault

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==Project Description/Abstract==
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