TX-TL projects, 2015-16 - Revision history

Chayes at 07:08, 27 January 2016

2016-01-27T07:08:38Z

← Older revision		Revision as of 07:08, 27 January 2016
Line 9:		Line 9:
	A number of projects are available for 2015-16 for undergraduates interested in contributing to the research progress in this area. Possible projects include:		A number of projects are available for 2015-16 for undergraduates interested in contributing to the research progress in this area. Possible projects include:
	* '''Variability in circuit performance across extract preparation methods.''' We have seen many situations in which circuits work only in batches of extract produced with certain methods of lysis (bead beating, homogenization, french press, etc). There are no obvious patterns of what types of circuits work in what types of extract, and so there are a lot of interesting speculation about what is going on. Work in this area would require testing existing circuits in different extract batches and then debugging the circuits in the batches where they don't work correctly.		* '''Variability in circuit performance across extract preparation methods.''' We have seen many situations in which circuits work only in batches of extract produced with certain methods of lysis (bead beating, homogenization, french press, etc). There are no obvious patterns of what types of circuits work in what types of extract, and so there are a lot of interesting speculation about what is going on. Work in this area would require testing existing circuits in different extract batches and then debugging the circuits in the batches where they don't work correctly.
	* '''Freeze-drying TX-TL.''' Currently, extract & buffer have to be stored at -80C, making transportation and distribution to non-science facilities difficult. One potential solution is to freeze-dry, or lyophilize, TX-TL, which may preserve activity at higher temperatures. Research will focus on determining optimal lyophilization & storage conditions for TX-TL, and may include trying vacuum sealing, stoppering chambers, or desiccant. Students will also test different types of circuits in TX-TL, to determine how lyophilization effects circuit function. This project may overlap with the Paper-based TX-TL project, as this research lyophilizing TX-TL onto paper.		* '''Freeze-drying TX-TL.''' Currently, extract & buffer have to be stored at -80C, making transportation and distribution to non-science facilities difficult. One potential solution is to freeze-dry, or lyophilize, TX-TL, which may preserve activity at higher temperatures. Research will focus on determining optimal lyophilization & storage conditions for TX-TL, and may include trying vacuum sealing, stoppering chambers, or desiccant. Students will also test different types of circuits in TX-TL, to determine how lyophilization effects circuit function. This project may overlap with the Paper-based TX-TL project, as this research includes lyophilizing TX-TL onto paper.
	* '''Paper-based TX-TL.''' Currently, TX-TL reactions are run either in plate wells or microcentrifuge tubes, with the fluorescent output of reactions measured in a plate reader. However, recently researchers in the Murray lab have found a way to measure the output of TX-TL reactions with a cellphone, if the TX-TL reactions are on paper. This project focuses on developing paper-based technology for TX-TL, with the aim of eventually being able to run all reactions on paper and read all outputs on a cellphone. Research will include designing paper reaction strips, testing different circuits on the strips, and modifying the cellphone to measure multiple fluorescent and luminescent reporter proteins. This project may overlap with the Freeze-drying TX-TL project, as it may involve lyophilizing TX-TL onto paper.		* '''Paper-based TX-TL.''' Currently, TX-TL reactions are run either in plate wells or microcentrifuge tubes, with the fluorescent output of reactions measured in a plate reader. However, recently researchers in the Murray lab have found a way to measure the output of TX-TL reactions with a cellphone, if the TX-TL reactions are on paper. This project focuses on developing paper-based technology for TX-TL, with the aim of eventually being able to run all reactions on paper and read all outputs on a cellphone. Research will include designing paper reaction strips, testing different circuits on the strips, and modifying the cellphone to measure multiple fluorescent and luminescent reporter proteins. This project may overlap with the Freeze-drying TX-TL project, as it may involve lyophilizing TX-TL onto paper.
	* '''Colorimetric reporter proteins.''' Currently, the outputs of TX-TL reactions are almost always fluorescent proteins, easy to express in TX-TL and commonly used in biology. However, special excitation-emission filters are needed to measure fluorescence, making it difficult to measure outside of a laboratory setting. Finding a TX-TL output that can be seen by the naked eye, eg. a colored protein or an enzyme-mediated color change, would help enable TX-TL use in the field. Research focuses on testing the functionality of different colorimetric reporters, the LacZ enzyme and varied LacZ substrates in TX-TL. This project may overlap with the Paper-based TX-TL project, as it may include testing successful colorimetric reporter proteins on paper.		* '''Colorimetric reporter proteins.''' Currently, the outputs of TX-TL reactions are almost always fluorescent proteins, easy to express in TX-TL and commonly used in biology. However, special excitation-emission filters are needed to measure fluorescence, making it difficult to measure outside of a laboratory setting. Finding a TX-TL output that can be seen by the naked eye, eg. a colored protein or an enzyme-mediated color change, would help enable TX-TL use in the field. Research focuses on testing the functionality of different colorimetric reporters, the LacZ enzyme and varied LacZ substrates in TX-TL. This project may overlap with the Paper-based TX-TL project, as it may include testing successful colorimetric reporter proteins on paper.
	* '''Improvements in preparation methods for TX-TL.''' Current preparation methods to make extract for circuit prototyping are low-yield (18 mL per batch). However, alternative preparation methods exist (45 mL per batch) which are significantly less labor-intensive, but are not optimized for circuit prototyping. Research on preparation methods will be conducted to increase yields but match cellular conditions more precisely.		* '''Improvements in preparation methods for TX-TL.''' Current preparation methods to make extract for circuit prototyping are low-yield (18 mL per batch). However, alternative preparation methods exist (45 mL per batch) which are significantly less labor-intensive, but are not optimized for circuit prototyping. Research on preparation methods will be conducted to increase yields but match cellular conditions more precisely.

Chayes at 07:08, 27 January 2016

2016-01-27T07:08:21Z

← Older revision		Revision as of 07:08, 27 January 2016
Line 9:		Line 9:
	A number of projects are available for 2015-16 for undergraduates interested in contributing to the research progress in this area. Possible projects include:		A number of projects are available for 2015-16 for undergraduates interested in contributing to the research progress in this area. Possible projects include:
	* '''Variability in circuit performance across extract preparation methods.''' We have seen many situations in which circuits work only in batches of extract produced with certain methods of lysis (bead beating, homogenization, french press, etc). There are no obvious patterns of what types of circuits work in what types of extract, and so there are a lot of interesting speculation about what is going on. Work in this area would require testing existing circuits in different extract batches and then debugging the circuits in the batches where they don't work correctly.		* '''Variability in circuit performance across extract preparation methods.''' We have seen many situations in which circuits work only in batches of extract produced with certain methods of lysis (bead beating, homogenization, french press, etc). There are no obvious patterns of what types of circuits work in what types of extract, and so there are a lot of interesting speculation about what is going on. Work in this area would require testing existing circuits in different extract batches and then debugging the circuits in the batches where they don't work correctly.
	* '''Freeze-drying TX-TL.''' Currently, extract & buffer have to be stored at -80C, making transportation and distribution to non-science facilities difficult. One potential solution is to freeze-dry, or lyophilize, TX-TL, which may preserve activity at higher temperatures. Research will focus on determining optimal lyophilization & storage conditions for TX-TL, and may include trying vacuum sealing, stoppering chambers, or desiccant. Students will also test different types of circuits in TX-TL, to determine how lyophilization effects circuit function. This project may overlap with the Paper-based TX-TL project, as this research ~~includes figuring out how to lyophilize~~ TX-TL onto paper.		* '''Freeze-drying TX-TL.''' Currently, extract & buffer have to be stored at -80C, making transportation and distribution to non-science facilities difficult. One potential solution is to freeze-dry, or lyophilize, TX-TL, which may preserve activity at higher temperatures. Research will focus on determining optimal lyophilization & storage conditions for TX-TL, and may include trying vacuum sealing, stoppering chambers, or desiccant. Students will also test different types of circuits in TX-TL, to determine how lyophilization effects circuit function. This project may overlap with the Paper-based TX-TL project, as this research lyophilizing TX-TL onto paper.
	* '''Paper-based TX-TL.''' Currently, TX-TL reactions are run either in plate wells or microcentrifuge tubes, with the fluorescent output of reactions measured in a plate reader. However, recently researchers in the Murray lab have found a way to measure the output of TX-TL reactions with a cellphone, if the TX-TL reactions are on paper. This project focuses on developing paper-based technology for TX-TL, with the aim of eventually being able to run all reactions on paper and read all outputs on a cellphone. Research will include designing paper reaction strips, testing different circuits on the strips, and modifying the cellphone to measure multiple fluorescent and luminescent reporter proteins. This project may overlap with the Freeze-drying TX-TL project, as it may involve lyophilizing TX-TL onto paper.		* '''Paper-based TX-TL.''' Currently, TX-TL reactions are run either in plate wells or microcentrifuge tubes, with the fluorescent output of reactions measured in a plate reader. However, recently researchers in the Murray lab have found a way to measure the output of TX-TL reactions with a cellphone, if the TX-TL reactions are on paper. This project focuses on developing paper-based technology for TX-TL, with the aim of eventually being able to run all reactions on paper and read all outputs on a cellphone. Research will include designing paper reaction strips, testing different circuits on the strips, and modifying the cellphone to measure multiple fluorescent and luminescent reporter proteins. This project may overlap with the Freeze-drying TX-TL project, as it may involve lyophilizing TX-TL onto paper.
	* '''Colorimetric reporter proteins.''' Currently, the outputs of TX-TL reactions are almost always fluorescent proteins, easy to express in TX-TL and commonly used in biology. However, special excitation-emission filters are needed to measure fluorescence, making it difficult to measure outside of a laboratory setting. Finding a TX-TL output that can be seen by the naked eye, eg. a colored protein or an enzyme-mediated color change, would help enable TX-TL use in the field. Research focuses on testing the functionality of different colorimetric reporters, the LacZ enzyme and varied LacZ substrates in TX-TL. This project may overlap with the Paper-based TX-TL project, as it may include testing successful colorimetric reporter proteins on paper.		* '''Colorimetric reporter proteins.''' Currently, the outputs of TX-TL reactions are almost always fluorescent proteins, easy to express in TX-TL and commonly used in biology. However, special excitation-emission filters are needed to measure fluorescence, making it difficult to measure outside of a laboratory setting. Finding a TX-TL output that can be seen by the naked eye, eg. a colored protein or an enzyme-mediated color change, would help enable TX-TL use in the field. Research focuses on testing the functionality of different colorimetric reporters, the LacZ enzyme and varied LacZ substrates in TX-TL. This project may overlap with the Paper-based TX-TL project, as it may include testing successful colorimetric reporter proteins on paper.
	* '''Improvements in preparation methods for TX-TL.''' Current preparation methods to make extract for circuit prototyping are low-yield (18 mL per batch). However, alternative preparation methods exist (45 mL per batch) which are significantly less labor-intensive, but are not optimized for circuit prototyping. Research on preparation methods will be conducted to increase yields but match cellular conditions more precisely.		* '''Improvements in preparation methods for TX-TL.''' Current preparation methods to make extract for circuit prototyping are low-yield (18 mL per batch). However, alternative preparation methods exist (45 mL per batch) which are significantly less labor-intensive, but are not optimized for circuit prototyping. Research on preparation methods will be conducted to increase yields but match cellular conditions more precisely.

Chayes at 07:07, 27 January 2016

2016-01-27T07:07:26Z

← Older revision		Revision as of 07:07, 27 January 2016
Line 8:		Line 8:

	A number of projects are available for 2015-16 for undergraduates interested in contributing to the research progress in this area. Possible projects include:		A number of projects are available for 2015-16 for undergraduates interested in contributing to the research progress in this area. Possible projects include:
	* Variability in circuit performance across extract preparation methods. We have seen many situations in which circuits work only in batches of extract produced with certain methods of lysis (bead beating, homogenization, french press, etc). There are no obvious patterns of what types of circuits work in what types of extract, and so there are a lot of interesting speculation about what is going on. Work in this area would require testing existing circuits in different extract batches and then debugging the circuits in the batches where they don't work correctly.		* '''Variability in circuit performance across extract preparation methods.''' We have seen many situations in which circuits work only in batches of extract produced with certain methods of lysis (bead beating, homogenization, french press, etc). There are no obvious patterns of what types of circuits work in what types of extract, and so there are a lot of interesting speculation about what is going on. Work in this area would require testing existing circuits in different extract batches and then debugging the circuits in the batches where they don't work correctly.
	* ~~Expression of membrane~~-~~bound proteins in~~ TX-TL. ~~Many circuit components require membrane bound proteins~~, ~~including ligand~~-~~binding proteins~~ and ~~light~~-~~responsive transcription factors~~. ~~Several of these components have been tested in~~ TX-TL ~~but have not yet shown~~ activity. ~~In addition~~, in ~~metabolic engineering there are several membrane bound components of core metabolic pathways that are currently not accessible for~~ TX-TL ~~based prototyping~~. ~~There are several promising approaches for integrating membrane~~-~~like structures into cell~~-~~free environments (). We will characterize several of these approaches and demonstrate one or more that can be used~~ to ~~express functional, membrane~~-~~associated proteins~~.		* '''Freeze-drying TX-TL.''' Currently, extract & buffer have to be stored at -80C, making transportation and distribution to non-science facilities difficult. One potential solution is to freeze-dry, or lyophilize, TX-TL, which may preserve activity at higher temperatures. Research will focus on determining optimal lyophilization & storage conditions for TX-TL, and may include trying vacuum sealing, stoppering chambers, or desiccant. Students will also test different types of circuits in TX-TL, to determine how lyophilization effects circuit function. This project may overlap with the Paper-based TX-TL project, as this research includes figuring out how to lyophilize TX-TL onto paper.
	* ~~Implementation of DNA editing machinery in~~ TX-TL. ~~Many groups~~ are ~~using DNA editing machinery as part~~ of ~~biological circuits~~. ~~The two most common are CRISPR/Cas9 and integrase/excisionase systems. The~~ Murray lab ~~has demonstrated that some integrase/excisionase systems~~ are ~~functional while others are not, and has also performed initial (so far unsuccessful) testing of CRISPR/Cas 9~~. ~~We plan to develop the protocols required to implement CRISPR~~-based ~~circuits as well as integrase~~-~~based circuits~~, ~~as well as explore~~ the ~~ability~~ of ~~these~~ circuits to ~~work on linear DNA (useful for rapid prototyping) versus plasmid DNA~~.		* '''Paper-based TX-TL.''' Currently, TX-TL reactions are run either in plate wells or microcentrifuge tubes, with the fluorescent output of reactions measured in a plate reader. However, recently researchers in the Murray lab have found a way to measure the output of TX-TL reactions with a cellphone, if the TX-TL reactions are on paper. This project focuses on developing paper-based technology for TX-TL, with the aim of eventually being able to run all reactions on paper and read all outputs on a cellphone. Research will include designing paper reaction strips, testing different circuits on the strips, and modifying the cellphone to measure multiple fluorescent and luminescent reporter proteins. This project may overlap with the Freeze-drying TX-TL project, as it may involve lyophilizing TX-TL onto paper.
	* Implementation of scaffold-~~based circuits using non~~-~~homologous kinases in~~ TX-TL. ~~The Murray lab has built several circuits that make use~~ of ~~scaffold~~-~~based kinases~~ to ~~implement feedback regulation strategies (Hsiao et al 2013). Repeated attempts at Caltech to implement these circuits~~ in TX-TL ~~have so far failed~~ to ~~create working circuits. By~~ making ~~use~~ of the ~~improved extracts in WP1 along with more sophisticated analysis techniques (~~eg, ~~micro~~-~~arrays~~, ~~fluorescent probes and fluorophore/quencher systems), we will debug these circuits~~ and ~~enable them to be used as components~~ in TX-TL ~~circuits~~.		* '''Colorimetric reporter proteins.''' Currently, the outputs of TX-TL reactions are almost always fluorescent proteins, easy to express in TX-TL and commonly used in biology. However, special excitation-emission filters are needed to measure fluorescence, making it difficult to measure outside of a laboratory setting. Finding a TX-TL output that can be seen by the naked eye, eg. a colored protein or an enzyme-mediated color change, would help enable TX-TL use in the field. Research focuses on testing the functionality of different colorimetric reporters, the LacZ enzyme and varied LacZ substrates in TX-TL. This project may overlap with the Paper-based TX-TL project, as it may include testing successful colorimetric reporter proteins on paper.
	* Improvements in preparation methods for TX-TL. Current preparation methods to make extract for circuit prototyping are low-yield (18 mL per batch). However, alternative preparation methods exist (45 mL per batch) which are significantly less labor-intensive, but are not optimized for circuit prototyping. Research on preparation methods will be conducted to increase yields but match cellular conditions more precisely.		* '''Improvements in preparation methods for TX-TL.''' Current preparation methods to make extract for circuit prototyping are low-yield (18 mL per batch). However, alternative preparation methods exist (45 mL per batch) which are significantly less labor-intensive, but are not optimized for circuit prototyping. Research on preparation methods will be conducted to increase yields but match cellular conditions more precisely.
	* Implementation of a TX-TL based educational model for high school students. Create a pilot lab that will allow students to build different types of two-input logic gates by combining different parts of DNA. By combining and selecting from 12 different DNA parts, any two-input logic gates can be created. Development of new methods for rapidly characterizing circuit performance using an inexpensive cell-phone based reader and paper-based expression.

Murray at 01:38, 1 December 2015

2015-12-01T01:38:47Z

← Older revision		Revision as of 01:38, 1 December 2015
Line 12:		Line 12:
	* Implementation of DNA editing machinery in TX-TL. Many groups are using DNA editing machinery as part of biological circuits. The two most common are CRISPR/Cas9 and integrase/excisionase systems. The Murray lab has demonstrated that some integrase/excisionase systems are functional while others are not, and has also performed initial (so far unsuccessful) testing of CRISPR/Cas 9. We plan to develop the protocols required to implement CRISPR-based circuits as well as integrase-based circuits, as well as explore the ability of these circuits to work on linear DNA (useful for rapid prototyping) versus plasmid DNA.		* Implementation of DNA editing machinery in TX-TL. Many groups are using DNA editing machinery as part of biological circuits. The two most common are CRISPR/Cas9 and integrase/excisionase systems. The Murray lab has demonstrated that some integrase/excisionase systems are functional while others are not, and has also performed initial (so far unsuccessful) testing of CRISPR/Cas 9. We plan to develop the protocols required to implement CRISPR-based circuits as well as integrase-based circuits, as well as explore the ability of these circuits to work on linear DNA (useful for rapid prototyping) versus plasmid DNA.
	* Implementation of scaffold-based circuits using non-homologous kinases in TX-TL. The Murray lab has built several circuits that make use of scaffold-based kinases to implement feedback regulation strategies (Hsiao et al 2013). Repeated attempts at Caltech to implement these circuits in TX-TL have so far failed to create working circuits. By making use of the improved extracts in WP1 along with more sophisticated analysis techniques (eg, micro-arrays, fluorescent probes and fluorophore/quencher systems), we will debug these circuits and enable them to be used as components in TX-TL circuits.		* Implementation of scaffold-based circuits using non-homologous kinases in TX-TL. The Murray lab has built several circuits that make use of scaffold-based kinases to implement feedback regulation strategies (Hsiao et al 2013). Repeated attempts at Caltech to implement these circuits in TX-TL have so far failed to create working circuits. By making use of the improved extracts in WP1 along with more sophisticated analysis techniques (eg, micro-arrays, fluorescent probes and fluorophore/quencher systems), we will debug these circuits and enable them to be used as components in TX-TL circuits.
			* Improvements in preparation methods for TX-TL. Current preparation methods to make extract for circuit prototyping are low-yield (18 mL per batch). However, alternative preparation methods exist (45 mL per batch) which are significantly less labor-intensive, but are not optimized for circuit prototyping. Research on preparation methods will be conducted to increase yields but match cellular conditions more precisely.
	~~More information on~~ TX-TL ~~is available here: http://www~~.~~openwetware~~.~~org/wiki/Biomolecular_Breadboards~~		* Implementation of a TX-TL based educational model for high school students. Create a pilot lab that will allow students to build different types of two-input logic gates by combining different parts of DNA. By combining and selecting from 12 different DNA parts, any two-input logic gates can be created. Development of new methods for rapidly characterizing circuit performance using an inexpensive cell-phone based reader and paper-based expression.

Murray at 15:42, 26 September 2015

2015-09-26T15:42:37Z

← Older revision		Revision as of 15:42, 26 September 2015
Line 8:		Line 8:

	A number of projects are available for 2015-16 for undergraduates interested in contributing to the research progress in this area. Possible projects include:		A number of projects are available for 2015-16 for undergraduates interested in contributing to the research progress in this area. Possible projects include:
			* Variability in circuit performance across extract preparation methods. We have seen many situations in which circuits work only in batches of extract produced with certain methods of lysis (bead beating, homogenization, french press, etc). There are no obvious patterns of what types of circuits work in what types of extract, and so there are a lot of interesting speculation about what is going on. Work in this area would require testing existing circuits in different extract batches and then debugging the circuits in the batches where they don't work correctly.
	* Expression of membrane-bound proteins in TX-TL. Many circuit components require membrane bound proteins, including ligand-binding proteins and light-responsive transcription factors. Several of these components have been tested in TX-TL but have not yet shown activity. In addition, in metabolic engineering there are several membrane bound components of core metabolic pathways that are currently not accessible for TX-TL based prototyping. There are several promising approaches for integrating membrane-like structures into cell-free environments (). We will characterize several of these approaches and demonstrate one or more that can be used to express functional, membrane-associated proteins.		* Expression of membrane-bound proteins in TX-TL. Many circuit components require membrane bound proteins, including ligand-binding proteins and light-responsive transcription factors. Several of these components have been tested in TX-TL but have not yet shown activity. In addition, in metabolic engineering there are several membrane bound components of core metabolic pathways that are currently not accessible for TX-TL based prototyping. There are several promising approaches for integrating membrane-like structures into cell-free environments (). We will characterize several of these approaches and demonstrate one or more that can be used to express functional, membrane-associated proteins.
	* Implementation of DNA editing machinery in TX-TL. Many groups are using DNA editing machinery as part of biological circuits. The two most common are CRISPR/Cas9 and integrase/excisionase systems. The Murray lab has demonstrated that some integrase/excisionase systems are functional while others are not, and has also performed initial (so far unsuccessful) testing of CRISPR/Cas 9. We plan to develop the protocols required to implement CRISPR-based circuits as well as integrase-based circuits, as well as explore the ability of these circuits to work on linear DNA (useful for rapid prototyping) versus plasmid DNA.		* Implementation of DNA editing machinery in TX-TL. Many groups are using DNA editing machinery as part of biological circuits. The two most common are CRISPR/Cas9 and integrase/excisionase systems. The Murray lab has demonstrated that some integrase/excisionase systems are functional while others are not, and has also performed initial (so far unsuccessful) testing of CRISPR/Cas 9. We plan to develop the protocols required to implement CRISPR-based circuits as well as integrase-based circuits, as well as explore the ability of these circuits to work on linear DNA (useful for rapid prototyping) versus plasmid DNA.

Murray: Created page with "{| style="float: right" |- | 400px |- | align=center | Overview of the cell-free expression breadboard process. |} The overall goal of this r..."

2015-08-28T04:50:11Z

Created page with "{| style="float: right" |- | 400px |- | align=center | Overview of the cell-free expression breadboard process. |} The overall goal of this r..."

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{| style="float: right"
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| [[Image:breadboards-process.png|400px]]
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| align=center | Overview of the cell-free expression breadboard process.
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The overall goal of this research is to build a set of “biomolecular breadboards” to create a systematic, engineering-oriented approach to synthesizing biomolecular circuits that involves developing, modeling, and debugging a sequence of prototype devices, each at increasing levels of complexity and each allowing the incorporation of increasingly realistic operating environments for either in vitro or in vivo applications. We are adapting an existing cell-free toolbox developed at U. Minnesota to create a set of prototyping environments for testing biological circuits. A sequence of increasingly complex environments, ending in prokaryotic cells, is being used to demonstrate the ability to prototype circuits that function in vivo, with iteration in in vitro assays and models to streamline development of predictable, in vivo functionality.

A number of projects are available for 2015-16 for undergraduates interested in contributing to the research progress in this area. Possible projects include:
* Expression of membrane-bound proteins in TX-TL. Many circuit components require membrane bound proteins, including ligand-binding proteins and light-responsive transcription factors. Several of these components have been tested in TX-TL but have not yet shown activity. In addition, in metabolic engineering there are several membrane bound components of core metabolic pathways that are currently not accessible for TX-TL based prototyping. There are several promising approaches for integrating membrane-like structures into cell-free environments (). We will characterize several of these approaches and demonstrate one or more that can be used to express functional, membrane-associated proteins.
* Implementation of DNA editing machinery in TX-TL. Many groups are using DNA editing machinery as part of biological circuits. The two most common are CRISPR/Cas9 and integrase/excisionase systems. The Murray lab has demonstrated that some integrase/excisionase systems are functional while others are not, and has also performed initial (so far unsuccessful) testing of CRISPR/Cas 9. We plan to develop the protocols required to implement CRISPR-based circuits as well as integrase-based circuits, as well as explore the ability of these circuits to work on linear DNA (useful for rapid prototyping) versus plasmid DNA.
* Implementation of scaffold-based circuits using non-homologous kinases in TX-TL. The Murray lab has built several circuits that make use of scaffold-based kinases to implement feedback regulation strategies (Hsiao et al 2013). Repeated attempts at Caltech to implement these circuits in TX-TL have so far failed to create working circuits. By making use of the improved extracts in WP1 along with more sophisticated analysis techniques (eg, micro-arrays, fluorescent probes and fluorophore/quencher systems), we will debug these circuits and enable them to be used as components in TX-TL circuits.

More information on TX-TL is available here: http://www.openwetware.org/wiki/Biomolecular_Breadboards