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A synthetic positive feedback based gene regulatory circuit

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Title: A synthetic positive feedback based gene regulatory circuit
Author(s): Nistala, Goutam J.
Director of Research: Bhalerao, Kaustubh D.
Doctoral Committee Chair(s): Bhalerao, Kaustubh D.
Doctoral Committee Member(s): Rao, Christopher V.; Vodkin, Lila O.; Gennis, Robert B.
Department / Program: Engineering Administration
Discipline: Agricultural & Biological Engr
Degree Granting Institution: University of Illinois at Urbana-Champaign
Degree: Ph.D.
Genre: Dissertation
Subject(s): Synthetic Biology Positive Feedback Gene Regulatory Circuit Single Cell Biosensor Amplification Sensitivity Memory
Abstract: The concept of reengineering and rewiring of pathways and gene regulatory networks for novel uses, and sometimes for mimicking natural systems for various uses in various disciplines gradually acquired the name “synthetic biology”. Synthetic biology, with foundations rooted in multiple disciplines like biological engineering, molecular biology, genetic engineering, and systems biology to name a few, is a new approach towards developing applications for solving current global problems in areas of food, agricultural, environmental, health (Weber et al., 2008) and alternative energy. A synthetic biology approach was used to develop a positive feedback base gene regulatory circuit in E.coli. Positive feedback is a common mechanism used in the regulation of many gene circuits as it can amplify the response to inducers and also generate binary outputs and hysteresis. In the context of electrical circuit design, positive feedback is most often employed in the design of amplifiers. Similar approaches may therefore be applied to design modular amplifier for the design of synthetic gene circuits and cell-based sensors. A modular positive feedback circuit was developed that can function as a genetic signal amplifier, heightening the sensitivity to inducer signals as well as increasing maximum expression levels without the need for an external cofactor. The design utilizes a constitutively active, autoinducer-independent variant of the quorum-sensing regulator LuxR. The ability of the positive feedback module to separately amplify the output of a one-component tetracycline sensor and a two-component aspartate sensor was experimentally tested and validated. In each case, the positive feedback module amplified the response to the respective inducers, both with regards to the gain and sensitivity. The advantage of this design is that the actual feedback mechanism depends only on a single gene and does not require any other modulation. Furthermore, this circuit can amplify any transcriptional signal, not just one encoded within the circuit or as inducer concentration. As our design is modular, it can potentially be used as a component in the design of more complex synthetic gene circuits.
Issue Date: 2011-01-14
URI: http://hdl.handle.net/2142/18368
Rights Information: Copyright 2010 Goutam J. Nistala
Date Available in IDEALS: 2011-01-14
Date Deposited: 2010-12
 

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