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Biomolecular Breadboards: Difference between revisions
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=== Cell-free circuit breadboard === | === Cell-free circuit breadboard === | ||
The cell-free circuit breadboard family is a sequence 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 cell-free circuit breadboard family is a sequence 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 is eliminated during the preparation. The resulting protein synthesis machinery is used to program cell-free TX-TL gene circuits in reactions of typical volume 10 $\mu$l. The gene circuits are engineered in the laboratory using standard molecular cloning techniques. | ||
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 energy molecules, 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. PCR products can be used with some preparation. | |||
* No antibiotic resistance markers are required, allowing plasmids using the same backbone to be included with different components. The concentrations of different plasmids can be modulated by changing the concentration of the plasmid. | |||
* Approximate cost: $0.03/ul. Typical reactions are 10-20 ul ($0.30-0.60 per reaction) | |||
=== DNA origami breadboard === | === DNA origami breadboard === | ||
