Modular control of multiple pathways using engineered orthogonal T7 polymerases
Author(s)Temme, Karsten; Hill, Rena; Segall-Shapiro, Thomas Hale; Moser, Felix; Voigt, Christopher A.
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Synthetic genetic sensors and circuits enable programmable control over the timing and conditions of gene expression. They are being increasingly incorporated into the control of complex, multigene pathways and cellular functions. Here, we propose a design strategy to genetically separate the sensing/circuitry functions from the pathway to be controlled. This separation is achieved by having the output of the circuit drive the expression of a polymerase, which then activates the pathway from polymerase-specific promoters. The sensors, circuits and polymerase are encoded together on a ‘controller’ plasmid. Variants of T7 RNA polymerase that reduce toxicity were constructed and used as scaffolds for the construction of four orthogonal polymerases identified via part mining that bind to unique promoter sequences. This set is highly orthogonal and induces cognate promoters by 8- to 75-fold more than off-target promoters. These orthogonal polymerases enable four independent channels linking the outputs of circuits to the control of different cellular functions. As a demonstration, we constructed a controller plasmid that integrates two inducible systems, implements an AND logic operation and toggles between metabolic pathways that change Escherichia coli green (deoxychromoviridans) and red (lycopene). The advantages of this organization are that (i) the regulation of the pathway can be changed simply by introducing a different controller plasmid, (ii) transcription is orthogonal to host machinery and (iii) the pathway genes are not transcribed in the absence of a controller and are thus more easily carried without invoking evolutionary pressure.
DepartmentMassachusetts Institute of Technology. Department of Biological Engineering
Nucleic Acids Research
Oxford University Press (OUP)
Temme, K. et al. “Modular Control of Multiple Pathways Using Engineered Orthogonal T7 Polymerases.” Nucleic Acids Research (2012). Copyright © 2012 Oxford University Press
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