2013
DOI: 10.1021/sb400028c
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Probing Cell-Free Gene Expression Noise in Femtoliter Volumes

Abstract: Cell-free systems offer a simplified and flexible context that enables important biological reactions while removing complicating factors such as fitness, division, and mutation that are associated with living cells. However, cell-free expression in unconfined spaces is missing important elements of expression in living cells. In particular, the small volume of living cells can give rise to significant stochastic effects, which are negligible in bulk cell-free reactions. Here, we confine cell-free gene express… Show more

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Cited by 45 publications
(55 citation statements)
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“…In this approach, commercially available cell-free transcription/translation systems (bacterial or mammalian) are freeze-dried onto paper or other porous substrates to create poised genetic regulatory networks that are stable for long-term storage at room temperature and are activated by rehydration. This work follows on other efforts in cell-free synthetic biology, which have provided important dynamic and mechanistic insight on gene regulatory networks and allowed for rapid "build-test" cycles for prototyping engineered gene circuits and biomanufacturing pathways (36)(37)(38)(39)(40)(41). The in vitro nature of these systems resolves the challenge faced by cell-based approaches of importing biomolecular components into the intracellular space, making these cell-free environments easily modified and excellent platforms for engineering.…”
Section: In Vitro Diagnosticsmentioning
confidence: 99%
“…In this approach, commercially available cell-free transcription/translation systems (bacterial or mammalian) are freeze-dried onto paper or other porous substrates to create poised genetic regulatory networks that are stable for long-term storage at room temperature and are activated by rehydration. This work follows on other efforts in cell-free synthetic biology, which have provided important dynamic and mechanistic insight on gene regulatory networks and allowed for rapid "build-test" cycles for prototyping engineered gene circuits and biomanufacturing pathways (36)(37)(38)(39)(40)(41). The in vitro nature of these systems resolves the challenge faced by cell-based approaches of importing biomolecular components into the intracellular space, making these cell-free environments easily modified and excellent platforms for engineering.…”
Section: In Vitro Diagnosticsmentioning
confidence: 99%
“…Recently cell-free systems have begun to gain a foothold as viable platforms for synthetic biology [8][9][10] . The appeal of such approaches is that they free synthetic biology from the resource sharing and 'extrinsic noise' that affects reaction dynamics in living cells.…”
Section: Introductionmentioning
confidence: 99%
“…Although our DNA template concentrations were in the range of low copy number plasmid concentrations in E. coli, RNA and protein concentrations were higher than average cellular concentrations. Due to the relatively large size of our nanoreactors (two orders of magnitude larger than the giant bacterium Epulopiscium) (32), stochastic processes may be difficult to study at the moment (33). It should however be feasible to scale down the 33-nL reactors by one to two orders of magnitude with existing microfabrication approaches (34) and to use E. coil RNAP instead of a phage RNAP.…”
Section: Discussionmentioning
confidence: 99%