Biomolecular Breadboards: Difference between revisions
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A ''biomolecular breadboard'' is a system that is designed to allow certain features of a circuit to be tested in a carefully controlled setting. These breadboards can be used to implement, debug, and characterize a wide variety of circuits, including both ''in vivo'' and ''in vitro'' devices. This page contains an overview of different biomolecular breadboards that are available. | |||
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The figure below provides an overview of the basic breadboarding process. At the left is a circuit that we wish to implement and transform into a cell or other bimolecular chassis. Rather than try to directly get the circuit working in the cell, which requires time consuming iterations and difficult debugging, we instead use a sequence of simpler test environments ("breadboards"), where we can do much more rapid iterations between experiments, modeling and design. | |||
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[ | [[Image:breadboard-process.png|750px]] | ||
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* [http://www.cds.caltech.edu/~murray/talks/breadboards_sb6.0-09Jul13.pdf SB 6.0 presentation], 09 Jul 2013 ([http://sb6.biobricks.org/digital-conference/?id=70021102 video], starting at ~1h:10m) | |||
=== TX-TL cell-free circuit breadboard === | |||
The cell-free circuit breadboard family is a collection of ''in vitro'' protocols that can be used to test transcription and translation (TX-TL) circuits in a set of systematically-constructed environments that explore different elements of the external conditions in which the circuits must operate. This breadboard is based on the work of Vincent Noireaux at U. Minnesota. The transcription and translation machineries are extracted from ''E. coli'' cells (Shin and Noireaux, 2010). The endogenous DNA and mRNA from the cells are eliminated during the preparation. The resulting protein synthesis machinery is used to program cell-free TX-TL gene circuits in reactions of 12uL. The gene circuits can engineered in the laboratory using standard molecular cloning techniques, but it is also possible to use PCR products (linear DNA), which substantially decreases the design cycle time. | |||
Breadboard features: | |||
* The cell-free expression breadboard uses the ''E. coli'' housekeeping transcriptional machinery (σ70 and core RNA polymerase) to express all the other transcription factors, therefore providing all of the ''E. coli'' DNA regulatory components. | |||
* The reaction buffer is entirely custom-made from pure chemicals. The concentration of ATP and other nucleotides, ions, amino acids, molecular crowding agents, and other chemicals can be adjusted, as well as the pH of the reaction, over a wide range of concentrations. | |||
* The breadboard can express proteins from either circular (plasmid) or linear DNA. | |||
* No antibiotic resistance markers are required, allowing multiple plasmids with similar origins of replication to be included. The concentrations of different plasmids can be independently modulated. | |||
* Approximate cost: $0.01/ul. Typical reactions are 12 ul ($0.126 per reaction) | |||
Papers describing the use of the TX-TL breadboard: | |||
* J. Shin and V. Noireaux, [http://pubs.acs.org/doi/abs/10.1021/sb200016s An E. coli cell-free expression toolbox: application to synthetic gene circuits and artificial cells]. ''ACS Synthetic Biology'', 1(1):29–41, 2012. | |||
* Z. Z. Sun, C. A. Hayes, J. Shin, F. Caschera, R. M. Murray, V. Noireaux, [http://www.jove.com/video/50762/protocols-for-implementing-an-escherichia-coli-based-tx-tl-cell-free Protocols for Implementing an Escherichia Coli Based TX-TL Cell-Free Expression System for Synthetic Biology]. ''Journal of Visualized Experiments'', 79; doi: 10.3791/507622013. | |||
* Z. Z. Sun, E. Yeung, C. A. Hayes, V. Noireaux and Richard M. Murray, [http://www.cds.caltech.edu/~murray/papers/sun+13-acs_synbio.html Linear DNA for rapid prototyping of synthetic biological circuits in an ''Escherichia coli'' based TX-TL cell-free system]. ''ACS Synthetic Biology'', 2013 (submitted). | |||
* M. K. Takahashi, C. A. Hayes, J. Chappell, Z. Z. Sun, R. M. Murray, V. Noireaux, J. B. Lucks, [http://biorxiv.org/content/early/2015/05/21/019620 Characterizing and Prototyping Genetic Networks with Cell-Free Transcription-Translation Reactions]. ''Methods'', 2015. | |||
=== TX-TL modeling library === | |||
Although not strictly breadboard, we are also developing a modeling library that is a companion to the breadboards above and allows simulation of circuits, including effects of resource limits. The TX-TL toolbox for MATLAB is a set of MATLAB functions that are designed to simplify the modeling of circuits used in the TX-TL cell free expression system. The source code for the toolbox is available via [http://sourceforge.net/projects/txtl/ SourceForge]. | |||
More information is available on the [[Biomolecular Breadboards:Models|Models tab]]. | |||
Latest revision as of 06:17, 4 September 2015
| Home | Protocols | DNA parts | Preliminary Data | Models | More Info |
A biomolecular breadboard is a system that is designed to allow certain features of a circuit to be tested in a carefully controlled setting. These breadboards can be used to implement, debug, and characterize a wide variety of circuits, including both in vivo and in vitro devices. This page contains an overview of different biomolecular breadboards that are available.
The figure below provides an overview of the basic breadboarding process. At the left is a circuit that we wish to implement and transform into a cell or other bimolecular chassis. Rather than try to directly get the circuit working in the cell, which requires time consuming iterations and difficult debugging, we instead use a sequence of simpler test environments ("breadboards"), where we can do much more rapid iterations between experiments, modeling and design.
- SB 6.0 presentation, 09 Jul 2013 (video, starting at ~1h:10m)
TX-TL cell-free circuit breadboard
The cell-free circuit breadboard family is a collection of in vitro protocols that can be used to test transcription and translation (TX-TL) circuits in a set of systematically-constructed environments that explore different elements of the external conditions in which the circuits must operate. This breadboard is based on the work of Vincent Noireaux at U. Minnesota. The transcription and translation machineries are extracted from E. coli cells (Shin and Noireaux, 2010). The endogenous DNA and mRNA from the cells are eliminated during the preparation. The resulting protein synthesis machinery is used to program cell-free TX-TL gene circuits in reactions of 12uL. The gene circuits can engineered in the laboratory using standard molecular cloning techniques, but it is also possible to use PCR products (linear DNA), which substantially decreases the design cycle time.
Breadboard features:
- The cell-free expression breadboard uses the E. coli housekeeping transcriptional machinery (σ70 and core RNA polymerase) to express all the other transcription factors, therefore providing all of the E. coli DNA regulatory components.
- The reaction buffer is entirely custom-made from pure chemicals. The concentration of ATP and other nucleotides, ions, amino acids, molecular crowding agents, and other chemicals can be adjusted, as well as the pH of the reaction, over a wide range of concentrations.
- The breadboard can express proteins from either circular (plasmid) or linear DNA.
- No antibiotic resistance markers are required, allowing multiple plasmids with similar origins of replication to be included. The concentrations of different plasmids can be independently modulated.
- Approximate cost: $0.01/ul. Typical reactions are 12 ul ($0.126 per reaction)
Papers describing the use of the TX-TL breadboard:
- J. Shin and V. Noireaux, An E. coli cell-free expression toolbox: application to synthetic gene circuits and artificial cells. ACS Synthetic Biology, 1(1):29–41, 2012.
- Z. Z. Sun, C. A. Hayes, J. Shin, F. Caschera, R. M. Murray, V. Noireaux, Protocols for Implementing an Escherichia Coli Based TX-TL Cell-Free Expression System for Synthetic Biology. Journal of Visualized Experiments, 79; doi: 10.3791/507622013.
- Z. Z. Sun, E. Yeung, C. A. Hayes, V. Noireaux and Richard M. Murray, Linear DNA for rapid prototyping of synthetic biological circuits in an Escherichia coli based TX-TL cell-free system. ACS Synthetic Biology, 2013 (submitted).
- M. K. Takahashi, C. A. Hayes, J. Chappell, Z. Z. Sun, R. M. Murray, V. Noireaux, J. B. Lucks, Characterizing and Prototyping Genetic Networks with Cell-Free Transcription-Translation Reactions. Methods, 2015.
TX-TL modeling library
Although not strictly breadboard, we are also developing a modeling library that is a companion to the breadboards above and allows simulation of circuits, including effects of resource limits. The TX-TL toolbox for MATLAB is a set of MATLAB functions that are designed to simplify the modeling of circuits used in the TX-TL cell free expression system. The source code for the toolbox is available via SourceForge.
More information is available on the Models tab.
