Engineering and applications of genetic circuits

Sayut, Daniel J.; Kambam, Pavan Kumar Reddy; Sun, Lianhong

doi:10.1039/b700547d

Cited by 23 publications

(19 citation statements)

References 56 publications

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“…Finally, this work and our previous reports demonstrated that the genetic screen and selection that we developed could be used to engineer the components of quorum-sensing systems to change various properties, and these engineered quorum-sensing systems are expected to be useful in synthetic biology and metabolic engineering. [33] Experimental Section Bacterial strains, media, chemicals and culture conditions: E. coli strain Top10F' was used for all experiments. Unless otherwise stated, all chemicals were purchased from Sigma-Aldrich (St Louis, MO, USA).…”

Section: Discussionmentioning

confidence: 99%

Altering the Substrate Specificity of RhlI by Directed Evolution

et al. 2009

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REDUCING VIRULENCE: RhlI catalyzes the synthesis of N-butanoyl homoserine lactone (BHL), with a minor product N-hexanoyl homoserine lactone (HHL). By using directed evolution and a genetic screen, RhlI has been engineered for enhanced production of both BHL and HHL at a similar level. Quorum sensing regulates biofilm formation and virulence factor production in the human opportunistic pathogen Pseudomonas aeruginosa. We used directed evolution to engineer RhlI, an enzyme in the RhlI-RhlR quorum-sensing system of P. aeruginosa, to alter its substrate specificity and gain insight into the molecular mechanisms of quorum sensing. By using a genetic screen, we identified a mutant with improved production of RhlI's two signaling molecules, N-butanoyl- and N-hexanoyl-homoserine lactone (BHL and HHL). In particular, production of BHL has been enhanced by more than two-fold, and the synthesis of HHL has been improved from an undetectable level to a level similar to BHL; this change indicates a significant change in substrate specificity. No significant change in the gene expression level was observed. Sequence alignments suggest that the mutations are most likely to facilitate interactions between the enzyme and the two acylated ACP substrates. This work also demonstrates that the genetic screen/selection should be useful in engineering additional quorum-sensing components.

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Section: Discussionmentioning

confidence: 99%

Altering the Substrate Specificity of RhlI by Directed Evolution

et al. 2009

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“…Synthetic biologists envision a future in which combinations of well-characterized sequence elements lead to predictable outcomes, enabling the rational design of biological circuits and novel metabolic pathways (Andrianantoandro et al 2006;Heinemann & Panke 2006;Drubin et al 2007;Sayut et al 2007;Tyo et al 2007). Attempts to characterize and employ sequences that control transcription, mRNA stability and the initiation of translation are underway in many synthetic biology laboratories (Yokobayashi et al 2002;Sprinzak & Elowitz 2005;Heinemann & Panke 2006;Dasika & Maranas 2008;Michalodimitrakis & Isalan 2009).…”

Section: Introductionmentioning

confidence: 99%

You're one in a googol: optimizing genes for protein expression

Welch

Villalobos

Gustafsson

et al. 2009

J. R. Soc. Interface.

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A vast number of different nucleic acid sequences can all be translated by the genetic code into the same amino acid sequence. These sequences are not all equally useful however; the exact sequence chosen can have profound effects on the expression of the encoded protein.Despite the importance of protein-coding sequences, there has been little systematic study to identify parameters that affect expression. This is probably because protein expression has largely been tackled on an ad hoc basis in many independent projects: once a sequence has been obtained that yields adequate expression for that project, there is little incentive to continue work on the problem. Synthetic biology may now provide the impetus to transform protein expression folklore into design principles, so that DNA sequences may easily be designed to express any protein in any system. In this review, we offer a brief survey of the literature, outline the major challenges in interpreting existing data and constructing robust design algorithms, and propose a way to proceed towards the goal of rational sequence engineering.

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“…Overcoming these problems to create functional circuits can require multiple rounds of rational design and extensive changes to the initial circuit, including the replacement of key components. As alternatives to rational design, combinatorial methods provide a way to rapidly generate circuit diversity (17) but are ultimately limited by the small number of well-classified genetic elements (32). Another alternative for the tuning of genetic circuits is the creation and screening of mutant libraries for the key circuit components to identify mutants with properties that mathematical models predict will result in desired behaviors (18,32).…”

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confidence: 99%

“…This method takes advantage of the ability of models to predict circuit behavior (16,20) but relies on the availability of mutant libraries for the circuit components. Directed evolution is a particularly useful technique for the generation of these mutant libraries as it allows for the rapid generation of diversity that can be used to overcome limited understanding of the complex series of interactions that occur between circuit components (32,41). Here we demonstrate the utility of mutant component libraries in the optimization of circuit function by using a simple mathematical model to identify a set of mutant LuxR proteins from an existing directed evolution library that result in enhancement of the logical responses of the AND gate.…”

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confidence: 99%

“…As alternatives to rational design, combinatorial methods provide a way to rapidly generate circuit diversity (17) but are ultimately limited by the small number of well-classified genetic elements (32). Another alternative for the tuning of genetic circuits is the creation and screening of mutant libraries for the key circuit components to identify mutants with properties that mathematical models predict will result in desired behaviors (18,32). This method takes advantage of the ability of models to predict circuit behavior (16,20) but relies on the availability of mutant libraries for the circuit components.…”

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confidence: 99%

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Construction and Enhancement of a Minimal Genetic AND Logic Gate

Sayut

Niu

Sun

2009

Appl Environ Microbiol

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The ability of genetic networks to integrate multiple inputs in the generation of cellular responses is critical for the adaptation of cellular phenotype to distinct environments and of great interest in the construction of complex artificial circuits. To develop artificial genetic circuits that can integrate intercellular signaling molecules and commonly used inducing agents, we have constructed an artificial genetic AND gate based on the P luxI quorum-sensing promoter and the lac repressor. The hybrid promoter exhibited reduced basal and induced expression levels but increased expression capacity, generating clear logical responses that could be described using a simple mathematical model. The model also predicted that the AND gate's logic could be improved by altering the properties of the LuxR transcriptional activator and, in particular, by increasing its rate of transcriptional activation. Following these predictions, we were able to improve the AND gate's logic by ϳ1.5-fold using a LuxR mutant library generated by directed evolution, providing the first example of the use of mutant transcriptional activators to improve the logic of a complex regulatory circuit. In addition, detailed characterizations of the AND gate's responses shed light on how LuxR, LacI, and RNA polymerase interact to activate gene expression.Genetic regulatory systems are often composed of coupled components that integrate multiple inputs to increase the specificity of environmental responses and program complex cellular behavior (6, 21). Depending on their complexity, these regulatory systems can include components that function at the transcriptional (38) and posttranscriptional (1, 39) levels. In many systems, these components act to perform logical functions that are roughly analogous to those performed by traditional logic gates in electrical circuits and can be described using Boolean operators such as AND and OR. The prevalence of these logic gates in genetic regulatory circuits has prompted the creation of artificial circuits demonstrating diverse logical functions both to understand how logical responses are derived in natural systems and as modules for the creation of artificial circuits with increased complexities (2,(26)(27)(28)35).Logical AND responses are particularly common in biological networks, and studies have identified various mechanisms by which cells implement AND logic (29, 31). As part of the effort to classify these natural genetic logic gates and to generate novel gates for use in artificial genetic circuits, a variety of logical AND gates have been constructed using unique regulatory mechanisms including chemical complementation (10), posttranscriptional regulation (2), and allosteric control (31). While the complexities of these systems vary greatly, it has also been shown that AND logic can be obtained in a minimal system consisting of an individual promoter regulated by two transcription factors (11). A benefit of this simplified architecture is that it should allow for the easy construction of novel AND...

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Engineering and applications of genetic circuits

Cited by 23 publications

References 56 publications

Altering the Substrate Specificity of RhlI by Directed Evolution

Altering the Substrate Specificity of RhlI by Directed Evolution

You're one in a googol: optimizing genes for protein expression

Construction and Enhancement of a Minimal Genetic AND Logic Gate

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