Construction and Enhancement of a Minimal Genetic AND Logic Gate

Abstract

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...

Directed evolution: an evolving and enabling synthetic biology tool

“…For example, Lou and coworkers were able to tune a complex push-on push-off switch, which responds to a UV input by synthesizing one of two fluorescent reporter genes, through the evolution of ribosome binding sites for two key circuit components [28]. Sayut and coworkers, on the other hand, evolved a transcriptional activator with the guidance of mathematical modeling to enhance the functionality of an AND logic gate [29], while An and Chin utilized the o-mRNA and o-ribosome components to create feed-forward loops for transcription and translation [30]. Beyond the tuning of gene circuitry components, directed evolution can also be used to alter the input and output behaviors of gene switches.…”

Recent advances and opportunities in synthetic logic gates engineering in living cells

“…In a relevant study, the synthetic genetic circuits have been constructed by integrating intercellular signalling molecule. It is based on Plux quorum-sensing promoter and the lac repressor (Sayut et al 2009). Bagh et al (2010) developed a minimal genetic device with AND gate function in E. coli.…”

Biocomputers: from test tubes to live cells

“…Promising results on implementing transcriptional logic in mammalian cells was shown by Fussenegger and colleagues89. More recently a modification of yeast tri-hybrid system was shown to implement complex logic using small molecules as inputs and a set of interacting proteins as mediators90; a two-input AND gate using a complex promoter has been shown in bacteria91. It is hard to tell whether the scarcity of reports is due to lack of trying or to experimental challenges.…”