Engineering the logical properties of a genetic AND gate.

Synthetic biology promises to enhance our ability to control biological systems by creating a systematic approach for the construction of genetic circuits that reliably program cellular function. As part of this approach, efficient methods are needed for the tuning of genetic circuits so as to allow for optimization of a design despite varying cellular contexts and incomplete understanding of in vivo biological interactions. Here we outline an optimization method that we have used to improve the logical responses of a genetic AND logic gate derived from components of the LuxI-LuxR bacterial quorum-sensing system. Basing our approach on the idea of evolutionary design, we improved the properties of our genetic AND logic gate by using directed evolution and a two-step screening process to alter the activities of the LuxR transcriptional activator. Using this method, we were able to rapidly enhance the AND gate's logical responses and have increased the specificities of these responses by ∼1.5-fold.

Creating designer laccases.

High redox potential laccases from white-rot fungi are recalcitrant to engineering. Maté et al. (2010) employed directed evolution to improve the activity and expression level of the fungal laccase from basidiomycete PM1, followed by rational design to restore thermostability lost during evolution, resulting in a highly active and stable enzyme.

Slow activator degradation reduces the robustness of a coupled feedback loop oscillator.

Genetic circuits composed of coupled positive and negative feedback loops have been shown to occur as common motifs in natural oscillatory networks. Recent work in synthetic biology has begun to demonstrate how the properties and architectures of these circuits affect their behavior. Expanding on this work, we constructed a new implementation of a common coupled feedback loop architecture by incorporating the LuxR transcriptional activator as the positive feedback element. We found that the properties of the LuxR activator had a significant impact on the observed behavior of the coupled feedback loop circuit, as a slow degradation rate of LuxR led to its accumulation after initial circuit induction. Due to this accumulation, the presence of feedback on LuxR did not greatly alter the oscillatory behavior of the circuit from a control consisting of an independent negative feedback loop, with both systems showing oscillatory responses in 30-40% of the measured cells and highly variable periods. While the oscillatory properties of individual cells were not influenced by induction levels, the percentage of cells that demonstrated oscillations was. Slight improvements to the initial responses of the coupled feedback loop circuit were also obtained by coexpression of the GroE chaperones due to improved LuxR folding. These findings illustrate the importance that positive feedback has on the tunability and robustness of coupled feedback loop oscillators, and improve our understanding of how the behavior of these systems is impacted upon by their components' properties.

Altering the substrate specificity of RhlI by directed evolution.

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.

Construction and enhancement of a minimal genetic and logic gate.

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 approximately 1.5-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.

Directed evolution of LuxI for enhanced OHHL production.

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.

Engineering and applications of genetic circuits.

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.

Noise and kinetics of LuxR positive feedback loops.

We have previously reported the design and construction of positive feedback loops (PFLs) based on the LuxI-LuxR quorum-sensing system that can be used as modular transcriptional regulatory units for the construction of complex artificial genetic circuits. Here, we characterize these PFLs using single-cell and dynamic induction studies to fully understand their behavior and facilitate their incorporation into novel networks. The LuxR PFLs had graded responses to the OHHL signal molecule with inductions developing over time, causing a lag in response compared to a non-feedback control. The properties of the PFLs could be altered using LuxR mutants with altered sensitivities without changing the inherent properties of the systems. Because of their high sensitivity and ability to establish intercellular signaling, the LuxR PFLs described in this work could be used as well-defined modules for the construction of artificial genetic circuits.

Construction and engineering of positive feedback loops.

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.