CH391L/S12/In vitro Selection - Revision history

Adam Meyer at 14:55, 12 February 2012

Adam Meyer — Sun, 12 Feb 2012 14:55:36 GMT

←Older revision		Revision as of 14:55, 12 February 2012
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	==Overview of ''in vitro'' selection==		==Overview of ''in vitro'' selection==
	''In vitro'' selection refers to the pursuit of a biopolymer (protein or nucleic acid) with a new, desired function. Whether beginning from scratch, or starting with an existing sequence, the first step is to create a diverse library of variants, each with a different "fitness" (with respect to the desired function). The next step to enrich the library for variants with a higher fitness, thus increasing the overall fitness of the pool. Often the most difficult facet of a selection is the confinement of function such that a fit variant increases its own representation, without non-specifically helping its neighbors. The last step of a "round" of selection is the amplification of the recovered product and the regeneration of starting material. This allows for further cycles of enrichment and increased fitness. After several rounds of selection, individual variants are assayed for the desired functionality.		''In vitro'' selection refers to the pursuit of a biopolymer (protein or nucleic acid) with a new, desired function. Whether beginning from scratch, or starting with an existing sequence, the first step is to create a diverse library of variants, each with a different "fitness" (with respect to the desired function). The next step to enrich the library for variants with a higher fitness, thus increasing the overall fitness of the pool. Often the most difficult facet of a selection is the confinement of function such that a fit variant increases its own representation, without non-specifically helping its neighbors. The last step of a "round" of selection is the amplification of the recovered product and the regeneration of starting material. This allows for further cycles of enrichment and increased fitness. After several rounds of selection, individual variants are assayed for the desired functionality.
-

	===Library Generation===		===Library Generation===

Adam Meyer at 14:55, 12 February 2012

Adam Meyer — Sun, 12 Feb 2012 14:55:14 GMT

←Older revision		Revision as of 14:55, 12 February 2012
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	[[Category:CH391L_S12]]		[[Category:CH391L_S12]]
	==Overview of ''in vitro'' selection==		==Overview of ''in vitro'' selection==
		+	''In vitro'' selection refers to the pursuit of a biopolymer (protein or nucleic acid) with a new, desired function. Whether beginning from scratch, or starting with an existing sequence, the first step is to create a diverse library of variants, each with a different "fitness" (with respect to the desired function). The next step to enrich the library for variants with a higher fitness, thus increasing the overall fitness of the pool. Often the most difficult facet of a selection is the confinement of function such that a fit variant increases its own representation, without non-specifically helping its neighbors. The last step of a "round" of selection is the amplification of the recovered product and the regeneration of starting material. This allows for further cycles of enrichment and increased fitness. After several rounds of selection, individual variants are assayed for the desired functionality.
		+

	===Library Generation===		===Library Generation===

Jeffrey E. Barrick at 16:04, 3 February 2012

Jeffrey E. Barrick — Fri, 03 Feb 2012 16:04:45 GMT

←Older revision		Revision as of 16:04, 3 February 2012
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		+	[[Category:CH391L_S12]]
	==Overview of ''in vitro'' selection==		==Overview of ''in vitro'' selection==

Adam Meyer: /* References */

Adam Meyer — Sun, 29 Jan 2012 23:41:48 GMT

References

←Older revision		Revision as of 23:41, 29 January 2012
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	==References==		==References==
	<biblio>		<biblio>
-	#Ellington1990 Ellington, A. D., & Szostak, J. W. (1990). In vitro selection of RNA molecules that bind specific ligands. Nature, 346(6287), 818–822. ~~pmid=1697402~~	+	#Ellington1990 pmid=1697402 Ellington, A. D., & Szostak, J. W. (1990). In vitro selection of RNA molecules that bind specific ligands. Nature, 346(6287), 818–822.
-	#Bartel1993 Bartel, D., & Szostak, J. (1993). Isolation of new ribozymes from a large pool of random sequences. Science, 261(5127), 1411-1418. ~~pmid=7690155~~	+	#Bartel1993 pmid=7690155 Bartel, D., & Szostak, J. (1993). Isolation of new ribozymes from a large pool of random sequences. Science, 261(5127), 1411-1418.
-	#Tawfik1998 Tawfik, D S, & Griffiths, A D. (1998). Man-made cell-like compartments for molecular evolution. Nature biotechnology, 16(7), 652-6. ~~pmid=9661199~~	+	#Tawfik1998 pmid=9661199 Tawfik, D S, & Griffiths, A D. (1998). Man-made cell-like compartments for molecular evolution. Nature biotechnology, 16(7), 652-6.
-	#Griffiths2003 Griffiths, Andrew D, & Tawfik, Dan S. (2003). Directed evolution of an extremely fast phosphotriesterase by in vitro compartmentalization. The EMBO journal, 22(1), 24-35. ~~pmid=12505981~~	+	#Griffiths2003 pmid=12505981 Griffiths, Andrew D, & Tawfik, Dan S. (2003). Directed evolution of an extremely fast phosphotriesterase by in vitro compartmentalization. The EMBO journal, 22(1), 24-35.
	</biblio>		</biblio>

Adam Meyer: /* FACS */

Adam Meyer — Sun, 29 Jan 2012 23:34:31 GMT

FACS

←Older revision		Revision as of 23:34, 29 January 2012
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	=====FACS=====		=====FACS=====
	[[image:CH391L_S12_Griffiths_2003_schema.png \| Griffiths and Tawfik, 2003. EMBO\| right \|thumb\|200px]]		[[image:CH391L_S12_Griffiths_2003_schema.png \| Griffiths and Tawfik, 2003. EMBO\| right \|thumb\|200px]]
-	For much of the history of ''in vitro'' selection of protein function, the selection was for affinity or DNA modification. ~~Griffith~~ and Tawfik<cite>Griffiths2003</cite> used ''in vitro'' compartmentalization and FACS to select for phosphotriesterase activity. In an elegant scheme, biotinylated template and biotinylated anti-HA antibody were attached to streptavidin beads and emulsified with cell lysate. Upon transcription and translation, the HA-phosphotriesterase protein bound to the antibody. The bead-template-antibody-enzyme complex is stable upon emulsion breaking, and is re-emulsified with biotinylate substrate molecules. Active enzyme converts substrate to product, and the product-biotin complex binds to the bead. The bead-template-antibody-enzyme-product complexes are stable upon breaking the emulsion and the complex is incubated with anti-product antibodies. Thus, beads harboring genes encoding active enzymes are preferentially labeled by the anti-product antibody. The use of fluorescent antibodies and FACS allows enrichment of fluorescent beads and therefore active genes.	+	For much of the history of ''in vitro'' selection of protein function, the selection was for affinity or DNA modification. Griffiths and Tawfik<cite>Griffiths2003</cite> used ''in vitro'' compartmentalization and FACS to select for phosphotriesterase activity. In an elegant scheme, biotinylated template and biotinylated anti-HA antibody were attached to streptavidin beads and emulsified with cell lysate. Upon transcription and translation, the HA-phosphotriesterase protein bound to the antibody. The bead-template-antibody-enzyme complex is stable upon emulsion breaking, and is re-emulsified with biotinylate substrate molecules. Active enzyme converts substrate to product, and the product-biotin complex binds to the bead. The bead-template-antibody-enzyme-product complexes are stable upon breaking the emulsion and the complex is incubated with anti-product antibodies. Thus, beads harboring genes encoding active enzymes are preferentially labeled by the anti-product antibody. The use of fluorescent antibodies and FACS allows enrichment of fluorescent beads and therefore active genes.
		+
	=====Host Growth=====		=====Host Growth=====
	A plasmid expressing some gene that confers a growth advantage to its host will become over-represented relative to plasmids containing less functional versions of the gene.		A plasmid expressing some gene that confers a growth advantage to its host will become over-represented relative to plasmids containing less functional versions of the gene.

Adam Meyer at 23:33, 29 January 2012

Adam Meyer — Sun, 29 Jan 2012 23:33:44 GMT

←Older revision		Revision as of 23:33, 29 January 2012
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	The easiest function to enrich for is affinity for a ligand. To select for binders (aptamers for nucleic acids; antibodies and others for proteins,) one exposes the pool of potential binders to a fixed ligand. The best binders affix to the ligand while weaker binders are washed away. Those that remain can be amplified for further round of selection.		The easiest function to enrich for is affinity for a ligand. To select for binders (aptamers for nucleic acids; antibodies and others for proteins,) one exposes the pool of potential binders to a fixed ligand. The best binders affix to the ligand while weaker binders are washed away. Those that remain can be amplified for further round of selection.

-	For nucleic acids, the binder itself can be subject to amplification, as it is both the information-carrying and function-carrying molecule. In Ellington and Szostak ~~(1990)~~<cite>Ellington1990</cite>, A DNA library was created such a T7 promoter drives expression of an N100 RNA pool, flanked by constant regions. The pool is passed through a column with a bound ligand. Species that bind the ligand are retained on the column and weaker binders are washed away. Binders are eluted from the column and amplified to begin a new round.	+	For nucleic acids, the binder itself can be subject to amplification, as it is both the information-carrying and function-carrying molecule. In Ellington and Szostak<cite>Ellington1990</cite>, A DNA library was created such a T7 promoter drives expression of an N100 RNA pool, flanked by constant regions. The pool is passed through a column with a bound ligand. Species that bind the ligand are retained on the column and weaker binders are washed away. Binders are eluted from the column and amplified to begin a new round.

	For protein binders, the scheme must include linking of the information-carrying nucleic acid to the function-carrying protein. Some examples of this linking are phage display, cell-surface display, and ribosome display.		For protein binders, the scheme must include linking of the information-carrying nucleic acid to the function-carrying protein. Some examples of this linking are phage display, cell-surface display, and ribosome display.
	=====Selective Amplification=====		=====Selective Amplification=====
	[[image:CH391L_S12_Bartel_1993_schema.png \| Bartel and Szostak, 1993. Science\| thumb\|right\|100px]]		[[image:CH391L_S12_Bartel_1993_schema.png \| Bartel and Szostak, 1993. Science\| thumb\|right\|100px]]
-	The successful selection of aptamers from random sequence and the existence of ribozymes led to Bartel and Szostak ~~(1993)~~<cite>Bartel1993</cite> to address whether ribozymes could be selected from random sequence as well. A large random RNA pool was created with a constant semi-hairpin region at the 5'end and a constant 3' primer binding region on the 3' end. The pool was incubated with a substrate oligonucleotide that could complete the semi-hairpin and also as a 5' primer binding region. Only RNA molecules capable of covalently linking the substrate to itself would have both primer binding sites and thus be available as a PCR template. Thus, active sequences are selectively amplified at the expense of inactive ones. The result was several ribozyme ligases capable of forming 5'-3' or 5'-2' phosphodiester bonds.	+	The successful selection of aptamers from random sequence and the existence of ribozymes led to Bartel and Szostak<cite>Bartel1993</cite> to address whether ribozymes could be selected from random sequence as well. A large random RNA pool was created with a constant semi-hairpin region at the 5'end and a constant 3' primer binding region on the 3' end. The pool was incubated with a substrate oligonucleotide that could complete the semi-hairpin and also as a 5' primer binding region. Only RNA molecules capable of covalently linking the substrate to itself would have both primer binding sites and thus be available as a PCR template. Thus, active sequences are selectively amplified at the expense of inactive ones. The result was several ribozyme ligases capable of forming 5'-3' or 5'-2' phosphodiester bonds.

	=====Protection=====		=====Protection=====
-	The first ''in vitro'' evolved protein functions involved modification of the nucleic acid species that encoded it. Perhaps the first such function was protection of the DNA template. In Tawfik and Griffiths ~~(1998)~~ The template encodes HaeIII methyl transferase. Upon transcription and translation in bacterial lysate, active methyltransferase methylate HaeIII recognition sequences in the gene. The methylated genes are then protected from digestion by HaeIII endonuclease. Undigested templates are then amplified before the next round. The key to this experiment is the use of ''in vitro'' compartmentalization (described below)which allow an active methyltransferase to methylate its parent template, but not other templates in the pool.	+	The first ''in vitro'' evolved protein functions involved modification of the nucleic acid species that encoded it. Perhaps the first such function was protection of the DNA template. In Tawfik and Griffiths<cite>Tawfik1998</cite> The template encodes HaeIII methyl transferase. Upon transcription and translation in bacterial lysate, active methyltransferase methylate HaeIII recognition sequences in the gene. The methylated genes are then protected from digestion by HaeIII endonuclease. Undigested templates are then amplified before the next round. The key to this experiment is the use of ''in vitro'' compartmentalization (described below)which allow an active methyltransferase to methylate its parent template, but not other templates in the pool.
	=====FACS=====		=====FACS=====
	[[image:CH391L_S12_Griffiths_2003_schema.png \| Griffiths and Tawfik, 2003. EMBO\| right \|thumb\|200px]]		[[image:CH391L_S12_Griffiths_2003_schema.png \| Griffiths and Tawfik, 2003. EMBO\| right \|thumb\|200px]]
-	For much of the history of ''in vitro'' selection of protein function, the selection was for affinity or DNA modification. Griffith and Tawfik ~~(2003)~~ used ''in vitro'' compartmentalization and FACS to select for phosphotriesterase activity. In an elegant scheme, biotinylated template and biotinylated anti-HA antibody were attached to streptavidin beads and emulsified with cell lysate. Upon transcription and translation, the HA-phosphotriesterase protein bound to the antibody. The bead-template-antibody-enzyme complex is stable upon emulsion breaking, and is re-emulsified with biotinylate substrate molecules. Active enzyme converts substrate to product, and the product-biotin complex binds to the bead. The bead-template-antibody-enzyme-product complexes are stable upon breaking the emulsion and the complex is incubated with anti-product antibodies. Thus, beads harboring genes encoding active enzymes are preferentially labeled by the anti-product antibody. The use of fluorescent antibodies and FACS allows enrichment of fluorescent beads and therefore active genes.	+	For much of the history of ''in vitro'' selection of protein function, the selection was for affinity or DNA modification. Griffith and Tawfik<cite>Griffiths2003</cite> used ''in vitro'' compartmentalization and FACS to select for phosphotriesterase activity. In an elegant scheme, biotinylated template and biotinylated anti-HA antibody were attached to streptavidin beads and emulsified with cell lysate. Upon transcription and translation, the HA-phosphotriesterase protein bound to the antibody. The bead-template-antibody-enzyme complex is stable upon emulsion breaking, and is re-emulsified with biotinylate substrate molecules. Active enzyme converts substrate to product, and the product-biotin complex binds to the bead. The bead-template-antibody-enzyme-product complexes are stable upon breaking the emulsion and the complex is incubated with anti-product antibodies. Thus, beads harboring genes encoding active enzymes are preferentially labeled by the anti-product antibody. The use of fluorescent antibodies and FACS allows enrichment of fluorescent beads and therefore active genes.
	=====Host Growth=====		=====Host Growth=====
	A plasmid expressing some gene that confers a growth advantage to its host will become over-represented relative to plasmids containing less functional versions of the gene.		A plasmid expressing some gene that confers a growth advantage to its host will become over-represented relative to plasmids containing less functional versions of the gene.
Line 54:		Line 54:
	#Ellington1990 Ellington, A. D., & Szostak, J. W. (1990). In vitro selection of RNA molecules that bind specific ligands. Nature, 346(6287), 818–822. pmid=1697402		#Ellington1990 Ellington, A. D., & Szostak, J. W. (1990). In vitro selection of RNA molecules that bind specific ligands. Nature, 346(6287), 818–822. pmid=1697402
	#Bartel1993 Bartel, D., & Szostak, J. (1993). Isolation of new ribozymes from a large pool of random sequences. Science, 261(5127), 1411-1418. pmid=7690155		#Bartel1993 Bartel, D., & Szostak, J. (1993). Isolation of new ribozymes from a large pool of random sequences. Science, 261(5127), 1411-1418. pmid=7690155
		+	#Tawfik1998 Tawfik, D S, & Griffiths, A D. (1998). Man-made cell-like compartments for molecular evolution. Nature biotechnology, 16(7), 652-6. pmid=9661199
		+	#Griffiths2003 Griffiths, Andrew D, & Tawfik, Dan S. (2003). Directed evolution of an extremely fast phosphotriesterase by in vitro compartmentalization. The EMBO journal, 22(1), 24-35. pmid=12505981
	</biblio>		</biblio>

Adam Meyer at 23:28, 29 January 2012

Adam Meyer — Sun, 29 Jan 2012 23:28:57 GMT

←Older revision		Revision as of 23:28, 29 January 2012
Line 26:		Line 26:
	The easiest function to enrich for is affinity for a ligand. To select for binders (aptamers for nucleic acids; antibodies and others for proteins,) one exposes the pool of potential binders to a fixed ligand. The best binders affix to the ligand while weaker binders are washed away. Those that remain can be amplified for further round of selection.		The easiest function to enrich for is affinity for a ligand. To select for binders (aptamers for nucleic acids; antibodies and others for proteins,) one exposes the pool of potential binders to a fixed ligand. The best binders affix to the ligand while weaker binders are washed away. Those that remain can be amplified for further round of selection.

-	For nucleic acids, the binder itself can be subject to amplification, as it is both the information-carrying and function-carrying molecule. In Ellington and Szostak (1990), A DNA library was created such a T7 promoter drives expression of an N100 RNA pool, flanked by constant regions. The pool is passed through a column with a bound ligand. Species that bind the ligand are retained on the column and weaker binders are washed away. Binders are eluted from the column and amplified to begin a new round.	+	For nucleic acids, the binder itself can be subject to amplification, as it is both the information-carrying and function-carrying molecule. In Ellington and Szostak (1990)<cite>Ellington1990</cite>, A DNA library was created such a T7 promoter drives expression of an N100 RNA pool, flanked by constant regions. The pool is passed through a column with a bound ligand. Species that bind the ligand are retained on the column and weaker binders are washed away. Binders are eluted from the column and amplified to begin a new round.

	For protein binders, the scheme must include linking of the information-carrying nucleic acid to the function-carrying protein. Some examples of this linking are phage display, cell-surface display, and ribosome display.		For protein binders, the scheme must include linking of the information-carrying nucleic acid to the function-carrying protein. Some examples of this linking are phage display, cell-surface display, and ribosome display.
	=====Selective Amplification=====		=====Selective Amplification=====
	[[image:CH391L_S12_Bartel_1993_schema.png \| Bartel and Szostak, 1993. Science\| thumb\|right\|100px]]		[[image:CH391L_S12_Bartel_1993_schema.png \| Bartel and Szostak, 1993. Science\| thumb\|right\|100px]]
-	The successful selection of aptamers from random sequence and the existence of ribozymes led to Bartel and Szostak (1993) to address whether ribozymes could be selected from random sequence as well. A large random RNA pool was created with a constant semi-hairpin region at the 5'end and a constant 3' primer binding region on the 3' end. The pool was incubated with a substrate oligonucleotide that could complete the semi-hairpin and also as a 5' primer binding region. Only RNA molecules capable of covalently linking the substrate to itself would have both primer binding sites and thus be available as a PCR template. Thus, active sequences are selectively amplified at the expense of inactive ones. The result was several ribozyme ligases capable of forming 5'-3' or 5'-2' phosphodiester bonds.	+	The successful selection of aptamers from random sequence and the existence of ribozymes led to Bartel and Szostak (1993)<cite>Bartel1993</cite> to address whether ribozymes could be selected from random sequence as well. A large random RNA pool was created with a constant semi-hairpin region at the 5'end and a constant 3' primer binding region on the 3' end. The pool was incubated with a substrate oligonucleotide that could complete the semi-hairpin and also as a 5' primer binding region. Only RNA molecules capable of covalently linking the substrate to itself would have both primer binding sites and thus be available as a PCR template. Thus, active sequences are selectively amplified at the expense of inactive ones. The result was several ribozyme ligases capable of forming 5'-3' or 5'-2' phosphodiester bonds.

	=====Protection=====		=====Protection=====
Line 52:		Line 52:
	==References==		==References==
	<biblio>		<biblio>
-	#1 Ellington, Szostak. 1990, pmid=1697402	+	#Ellington1990 Ellington, A. D., & Szostak, J. W. (1990). In vitro selection of RNA molecules that bind specific ligands. Nature, 346(6287), 818–822. pmid=1697402
-	#2 Bartel, D., & Szostak, J. (1993). Isolation of new ribozymes from a large pool of random sequences. Science, 261(5127), 1411-1418 pmid=7690155	+	#Bartel1993 Bartel, D., & Szostak, J. (1993). Isolation of new ribozymes from a large pool of random sequences. Science, 261(5127), 1411-1418. pmid=7690155
	</biblio>		</biblio>

Adam Meyer at 23:24, 29 January 2012

Adam Meyer — Sun, 29 Jan 2012 23:24:18 GMT

←Older revision		Revision as of 23:24, 29 January 2012
Line 51:		Line 51:

	==References==		==References==
		+	<biblio>
		+	#1 Ellington, Szostak. 1990, pmid=1697402
		+	#2 Bartel, D., & Szostak, J. (1993). Isolation of new ribozymes from a large pool of random sequences. Science, 261(5127), 1411-1418 pmid=7690155
		+	</biblio>

Adam Meyer: /* In vitro Compartmentalization */

Adam Meyer — Sun, 29 Jan 2012 23:09:03 GMT

In vitro Compartmentalization

←Older revision		Revision as of 23:09, 29 January 2012
Line 48:		Line 48:

	=====''In vitro'' Compartmentalization=====		=====''In vitro'' Compartmentalization=====
-	Mechanical mixing of water in oil and surfactant leads to an emulsion of stable water droplets. The size of the droplets is inversely proportional to the amount of energy put into the system. The compartments prevent functional molecules from affecting the templates in other compartments.	+	Mechanical mixing of water in oil and surfactant leads to an emulsion of stable water droplets in a continuous oil phase. The size of the droplets is inversely proportional to the amount of energy put into the system. The compartments prevent functional molecules from affecting the templates in other compartments.

	==References==		==References==

Adam Meyer: /* In vitro Compartmentalization */

Adam Meyer — Sun, 29 Jan 2012 23:08:26 GMT

In vitro Compartmentalization

←Older revision		Revision as of 23:08, 29 January 2012
Line 48:		Line 48:

	=====''In vitro'' Compartmentalization=====		=====''In vitro'' Compartmentalization=====
		+	Mechanical mixing of water in oil and surfactant leads to an emulsion of stable water droplets. The size of the droplets is inversely proportional to the amount of energy put into the system. The compartments prevent functional molecules from affecting the templates in other compartments.

	==References==		==References==