Biomolecular Breadboards: Difference between revisions

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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)
* [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)


=== Cell-free circuit breadboard ===
=== 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.
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.
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* 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).
* 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.
* 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.
=== Artificial cell breadboard ===
The artificial cell breadboard family consists of phospholipid vesicles that contain the TX-TL cell-free expression system.  Vesicles can range in size from 1 to 50 um in diameter.  We have demonstrated the ability to implement TX-TL circuits in vesicles and to use external inducers that diffuse through the vesicle membrane to trigger reactions inside vesicles.
=== DNA origami breadboard ===
A third breadboard family that we are developing explores the role of spatial localization and single molecule effects. For this, we are using
DNA origami to spatially organize, study, and optimize existing ''in vitro'' transcription circuits such as oscillators.  There are two kinds of spatial
organization that can be effected by DNA origami. The first kind of organization, ''local patterning'' is intrinsic to the DNA origami themselves. For example, a rectangular origami provides a roughly 100~nm x 70~nm surface onto which other molecules can be positioned with a resolution of about 6 nanometers.
A second kind of spatial organization afforded by DNA orgami is that of ''global placement''.  We are developing a method for placing individual DNA origami at lithographically-patterned sites on a silicon dioxide surface: sticky patches in the shape of DNA origami are created by e-beam, origami are washed over the surface, and they bind and orient on the sticky patches.  Because of the regular placement of origami, we expect to eventually be able to observe up to 1600 (40 x 40) origami localized circuits in parallel to achieve good statistics.


=== TX-TL modeling library ===
=== 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.
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.
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