Artificial positive feedback loops (PFLs) have been used as genetic amplifiers for enhancing the responses of weak promoters and in the creation of eukaryotic gene switches. Here we describe the construction and directed evolution of two PFLs based on the LuxR transcriptional activator and its cognate promoter, P luxI . The wild-type PFLs are completely activated by 10 nM of 3-oxo-hexanoyl-homoserine lactone (OHHL). Directed evolution of LuxR increased the sensitivity of the feedback loops, resulting in systems that are completely activated at OHHL concentrations of 5 nM, or approximately 3 molecules per cell. The responses of the PFLs can also be modulated by adjusting inducer concentrations. These highly sensitive yet regulatable PFLs can be used to construct larger artificial genetic networks to gain understanding of the design principles of complex biological systems and are expected to find various applications in industrial fermentation and gene therapy.
Quorum sensing is a common mechanism used by bacteria to coordinate population behavior, and is involved in a variety of biological processes, such as bioluminescence, virulence factor synthesis, antibiotic production, and biofilm formation. To engineer the LuxI enzyme of the LuxI-LuxR quorum-sensing system, we developed a high throughput genetic selection to identify LuxI mutants with improved OHHL (3-oxo-hexanoyl homoserine lactone) synthesis in E. coli. Using this genetic selection, we created LuxI mutants with improved OHHL synthesis rates and yields through directed evolution, identifying three LuxI mutants after two generations. An in vivo semi-quantitative method allowed for verification of the genetic screen and OHHL yields were quantified using HPLC-MS/MS, revealing an 80-fold increase in a mutant culture compared to the wildtype culture. In addition to OHHL, the yields of C6HSL (hexanoyl homoserine lactone) and C8HSL (octanoyl homoserine lactone) were also improved, and a slight change in substrate specificity towards C6HSL production was observed. Based on alignment with the crystal structure of EsaI, a homolog of LuxI, two mutations are most likely involved in enhancing the interactions between the enzyme and the substrates. The high throughput genetic selection and the semi-quantitative method can be conveniently modified for the directed evolution of LuxI homologs. The identification of these LuxI mutants has implications in synthetic biology, where they can be used for the construction of artificial genetic circuits. In addition, development of drugs that specifically target quorum sensing to attenuate the pathogenesis of gram-negative infectious bacteria might also benefit from the insights into the molecular mechanism of quorum sensing revealed by the amino acid substitutions.
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...
In the field of synthetic biology, recent genetic engineering efforts have enabled the construction of novel genetic circuits with diverse functionalities and unique activation mechanisms. Because of these advances, artificial genetic networks are becoming increasingly complex, and are demonstrating more robust behaviors with reduced crosstalk between defined modules. These properties have allowed for the identification of a growing set of design principles that govern genetic networks, and led to an increased number of applications for genetic circuits in the fields of metabolic engineering and biomedical engineering. Such progress indicates that synthetic biology is rapidly evolving into an integrated engineering practice that uses rational and combinatorial design of synthetic gene networks to solve complex problems in biology, medicine, and human health.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.