Directed evolution of RhlI to generate new and increased quorum sensing signal molecule catalytic activities.

Quorum sensing is a population density-dependent gene expression regulation mechanism in bacteria. The substrate specificity of RhlI, an enzyme in the RhlI-RhlR quorum sensing system of Pseudomonas aeruginosa, was explored by directed evolution to gain insight into the molecular mechanisms of quorum sensing. RhlI catalyzes S-adenosyl methionine and butanoyl or hexanoyl acyl carrier protein to form N-butanoyl homoserine lactone (BHL) and or N-hexanoyl homoserine lactone (HHL), respectively, none of which contain 3-oxo groups. We developed high-throughput genetic screening and selection methods to identify RhlI mutants via four rounds of directed evolution and identified RhlI-4M1 as the mutant that generated new catalytic activity and synthesized 3-oxo-hexanoyl homoserine lactone (OHHL) containing the 3-oxo group in Escherichia coli. Additionally, the synthesizing activities of BHL and HHL were improved by 3.98- and 3.01-fold, respectively. RhlI-4M1 contains five amino acid substitutions (A15D, K31R, T92S, Y129N, and L184Q) and one stop codon (Q193*) mutations. The deletion of nine amino acids in the C-terminus was crucial for OHHL production by RhlI mutants. This work demonstrates that the genetic screen/selection should be useful in the development of applications involving the manipulation of bacterial quorum sensing. The new catalytic activity of these RhlI mutants will prove beneficial in elucidating the mechanistic understanding of bacterial quorum sensing and similarly, may prove beneficial in the development of new drugs including antimicrobial compounds.

Directed evolution to improve the catalytic efficiency of urate oxidase from Bacillus subtilis.

Urate oxidase is a key enzyme in purine metabolism and catalyzes the oxidation of uric acid to allantoin. It is used to treat hyperuricemia and gout, and also in a diagnostic kit. In this study, error-prone polymerase chain reaction and staggered extension process was used to generate a mutant urate oxidase with improved enzyme activity from Bacillus subtilis. After several rounds of mutagenesis and screening, two mutants 6E9 and 8E279 were obtained which exhibited 2.99 and 3.43 times higher catalytic efficiency, respectively. They also exhibited lower optimal reaction temperature and higher thermo-stability. D44V, Q268R and K285Q were identified as the three most beneficial amino acid substitutions introduced by site-directed mutagenesis. D44V/Q268R, which was obtained through random combination of the three mutants, displayed the highest catalytic activity. The Km, kcat/Km and enzyme activity of D44V/Q268R increased by 68%, 83% and 129% respectively, compared with that of wild-type urate oxidase. Structural modeling indicated that mutations far from the active site can have significant effects on activity. For many of them, the underlying mechanisms are still difficult to explain from the static structural model. We also compared the effects of the same set of single point mutations on the wild type and on the final mutant. The results indicate strong effects of epistasis, which may imply that the mutations affect catalysis through influences on protein dynamics besides equilibrium structures.

Metabolic engineering of indole pyruvic acid biosynthesis in Escherichia coli with tdiD.

BACKGROUND: Indole pyruvic acid (IPA) is a versatile platform intermediate and building block for a number of high-value products in the pharmaceutical and food industries. It also has a wide range of applications, such as drugs for the nervous system, cosmetics, and luminophores. Chemical synthesis of IPA is a complicated and costly process. Moreover, through the biosynthesis route employing L-amino acid oxidase, the byproduct hydrogen peroxide leads the degradation of IPA. TdiD, identified as a specific tryptophan aminotransferase, could be an alternative solution for efficient IPA biosynthesis. RESULTS: Escherichia coli strain W3110, which demonstrates basic production when supplied with tryptophan, was engineered for IPA biosynthesis. Several strategies were implemented to improve IPA production. First, through incorporating the codon-optimized tdiD into W3110, IPA levels increased from 41.54 ± 1.26 to 52.54 ± 2.08 mg/L. Second, after verifying the benefit of an increased phenylpyruvate pool, a YL03 strain was constructed based on a previously reported mutant strain of W3110 with a plasmid carrying aroF fbr and pheA fbr to further improve IPA production. The recombinant YL03 strain accumulated IPA at 158.85 ± 5.36 mg/L, which was 3.82-fold higher than that of the wild-type W3110 strain. Third, optimization of tdiD co expression was carried out by replacing the Trc promoter with a series of constitutively active promoters along with increasing the plasmid copy numbers. The highest IPA production was observed in YL08, which achieved 236.42 ± 17.66 mg/L and represented a greater than 5-fold increase as compared to W3110. Finally, the effects of deletion and overexpression of tnaA on IPA biosynthesis were evaluated. The removal of tnaA led to slightly reduced IPA levels, whereas the overexpression of tnaA resulted in a considerable decline in production. CONCLUSIONS: This study illustrates the feasibility of IPA biosynthesis in E. coli through tdiD. An efficient IPA producing strain, YL08, was developed, which provides a new possibility for biosynthesis of IPA. Although the final production was limited, this study demonstrates a convenient method of IPA synthesis.

Engineered Kluyveromyces marxianus for pyruvate production at elevated temperature with simultaneous consumption of xylose and glucose.

Xylose and glucose from lignocellulose are sustainable sources for production of pyruvate, which is the starting material for the synthesis of many drugs and agrochemicals. In this study, the pyruvate decarboxylase gene (KmPDC1) and glycerol-3-phosphate dehydrogenase gene (KmGPD1) of Kluyveromyces marxianus YZJ051 were disrupted to prevent ethanol and glycerol accumulation. The deficient growth of PDC disruption was rescued by overexpressing mutant KmMTH1-ΔT. Then pentose phosphate pathway and xylitol dehydrogenase SsXYL2-ARS genes were overexpressed to obtain strain YZB053 which produced pyruvate with xylose other than glucose. It produced 24.62g/L pyruvate from 80g/L xylose with productivity of 0.51g/L/h at 42°C. Then, xylose-specific transporter ScGAL2-N376F was overexpressed to obtain strain YZB058, which simultaneously consumed 40g/L glucose and 20g/L xylose and produced 29.21g/L pyruvate with productivity of 0.81g/L/h at 42°C. Therefore, a platform for pyruvate production from glucose and xylose at elevated temperature was developed.

Simultaneous fermentation of glucose and xylose at elevated temperatures co-produces ethanol and xylitol through overexpression of a xylose-specific transporter in engineered Kluyveromyces marxianus.

Engineered Kluyveromyces marxianus strains were constructed through over-expression of various transporters for simultaneous co-fermentation of glucose and xylose. The glucose was converted into ethanol, whereas xylose was converted into xylitol which has higher value than ethanol. Over-expressing xylose-specific transporter ScGAL2-N376F mutant enabled yeast to co-ferment glucose and xylose and the co-fermentation ability was obviously improved through increasing ScGAL2-N376F expression. The production of glycerol was blocked and acetate production was reduced by disrupting gene KmGPD1. The obtained K. marxianus YZJ119 utilized 120g/L glucose and 60g/L xylose simultaneously and produced 50.10g/L ethanol and 55.88g/L xylitol at 42°C. The yield of xylitol from consumed xylose was over 98% (0.99g/g). Through simultaneous saccharification and co-fermentation at 42°C, YZJ119 produced a maximal concentration of 44.58g/L ethanol and 32.03g/L xylitol or 29.82g/L ethanol and 31.72g/L xylitol, respectively, from detoxified or non-detoxified diluted acid pretreated corncob.

Data for rapid ethanol production at elevated temperatures by engineered thermotolerant Kluyveromyces marxianus via the NADP(H)-preferring xylose reductase-xylitol dehydrogenase pathway.

A thermo-tolerant NADP(H)-preferring xylose pathway was constructed in Kluyveromyces marxianus for ethanol production with xylose at elevated temperatures (Zhang et al., 2015 [25]). Ethanol production yield and efficiency was enhanced by pathway engineering in the engineered strains. The constructed strain, YZJ088, has the ability to co-ferment glucose and xylose for ethanol and xylitol production, which is a critical step toward enabling economic biofuel production from lignocellulosic biomass. This study contains the fermentation results of strains using the metabolic pathway engineering procedure. The ethanol-producing abilities of various yeast strains under various conditions were compared, and strain YZJ088 showed the highest production and fastest productivity at elevated temperatures. The YZJ088 xylose fermentation results indicate that it fermented well with xylose at either low or high inoculum size. When fermented with an initial cell concentration of OD600=15 at 37 °C, YZJ088 consumed 200 g/L xylose and produced 60.07 g/L ethanol; when the initial cell concentration was OD600=1 at 37 °C, YZJ088 consumed 98.96 g/L xylose and produced 33.55 g/L ethanol with a productivity of 0.47 g/L/h. When fermented with 100 g/L xylose at 42 °C, YZJ088 produced 30.99 g/L ethanol with a productivity of 0.65 g/L/h, which was higher than that produced at 37 °C.

Rapid ethanol production at elevated temperatures by engineered thermotolerant Kluyveromyces marxianus via the NADP(H)-preferring xylose reductase-xylitol dehydrogenase pathway.

Conversion of xylose to ethanol by yeasts is a challenge because of the redox imbalances under oxygen-limited conditions. The thermotolerant yeast Kluyveromyces marxianus grows well with xylose as a carbon source at elevated temperatures, but its xylose fermentation ability is weak. In this study, a combination of the NADPH-preferring xylose reductase (XR) from Neurospora crassa and the NADP(+)-preferring xylitol dehydrogenase (XDH) mutant from Scheffersomyces stipitis (Pichia stipitis) was constructed. The xylose fermentation ability and redox balance of the recombinant strains were improved significantly by over-expression of several downstream genes. The intracellular concentrations of coenzymes and the reduced coenzyme/oxidized coenzyme ratio increased significantly in these metabolic strains. The byproducts, such as glycerol and acetic acid, were significantly reduced by the disruption of glycerol-3-phosphate dehydrogenase (GPD1). The resulting engineered K. marxianus YZJ088 strain produced 44.95 g/L ethanol from 118.39 g/L xylose with a productivity of 2.49 g/L/h at 42 °C. Additionally, YZJ088 realized glucose and xylose co-fermentation and produced 51.43 g/L ethanol from a mixture of 103.97 g/L xylose and 40.96 g/L glucose with a productivity of 2.14 g/L/h at 42 °C. These promising results validate the YZJ088 strain as an excellent producer of ethanol from xylose through the synthetic xylose assimilation pathway.

Protein-selective coacervation with hyaluronic acid.

Selective coacervation with hyaluronic acid (HA), a biocompatible and injectable anionic polysaccharide, was used to isolate a target protein, bovine serum albumin (BSA), with 90% purity from a 1:1 mixture with a second protein of similar pI, ß-lactoglobulin (BLG). This separation was attributed to the higher HA-affinity of BSA, arising from its more concentrated positive domain. The values of pH corresponding respectively to the onset of complex formation, coacervation, precipitation, and redissolution (pH(c), pHϕ, pH(p), and pH(d)) were determined as a function of ionic strength I. These pH values were related to critical values of protein charge, Z, and their dependence on I provided some insights into the mechanisms of these transitions. The higher polyanion binding affinity of BSA, deduced from its higher values of pH(c), was confirmed by isothermal titration calorimetry (ITC). Confocal laser microscopy clearly showed time-dependent coalescence of vesicular droplets into a continuous film. Comparisons with prior results for the polycation poly(diallyldimethylammonium chloride) (PDADMAC) show reversal of protein selectivity due to reversal of the polyelectrolyte charge. Stronger binding of both proteins to PDADMAC established by ITC may be related to the higher chain flexibility and effective linear charge density of this polycation.

Effect of heparin on protein aggregation: inhibition versus promotion.

The effect of heparin on both native and denatured protein aggregation was investigated by turbidimetry and dynamic light scattering (DLS). Turbidimetric data show that heparin is capable of inhibiting and reversing the native aggregation of bovine serum albumin (BSA), ß-lactoglobulin (BLG), and Zn-insulin at a pH near pI and at low ionic strength I; however, the results vary with regard to the range of pH, I, and protein-heparin stoichiometry required to achieve these effects. The kinetics of this process were studied to determine the mechanism by which interaction with heparin could result in inhibition or reversal of native protein aggregates. For each protein, the binding of heparin to distinctive intermediate aggregates formed at different times in the aggregation process dictates the outcome of complexation. This differential binding was explained by changes in the affinity of a given protein for heparin, partly due to the effects of protein charge anisotropy as visualized by electrostatic modeling. The heparin effect can be further extended to include inhibition of denaturing protein aggregation, as seen from the kinetics of BLG aggregation under conditions of thermally induced unfolding with and without heparin.

Multimerization and aggregation of native-state insulin: effect of zinc.

The aggregation of insulin is complicated by the coexistence of various multimers, especially in the presence of Zn(2+). Most investigations of insulin multimerization tend to overlook aggregation kinetics, while studies of insulin aggregation generally pay little attention to multimerization. A clear understanding of the starting multimer state of insulin is necessary for the elucidation of its aggregation mechanism. In this work, the native-state aggregation of insulin as either the Zn-insulin hexamer or the Zn-free dimer was studied by turbidimetry and dynamic light scattering, at low ionic strength and pH near pI. The two states were achieved by varying the Zn(2+) content of insulin at low concentrations, in accordance with size-exclusion chromatography results and literature findings (Tantipolphan, R.; Romeijn, S.; Engelsman, J. d.; Torosantucci, R.; Rasmussen, T.; Jiskoot, W. J. Pharm. Biomed. 2010, 52, 195). The much greater aggregation rate and limiting turbidity (τ(∞)) for the Zn-insulin hexamer relative to the Zn-free dimer was explained by their different aggregation mechanisms. Sequential first-order kinetic regimes and the concentration dependence of τ(∞) for the Zn-insulin hexamer indicate a nucleation and growth mechanism, as proposed by Wang and Kurganov (Wang, K.; Kurganov, B. I. Biophys. Chem. 2003, 106, 97). The pure second-order process for the Zn-free dimer suggests isodesmic aggregation, consistent with the literature. The aggregation behavior at an intermediate Zn(2+) concentration appears to be the sum of the two processes.

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.

Electrostatic selectivity in protein-nanoparticle interactions.

The binding of bovine serum albumin (BSA) and ß-lactoglobulin (BLG) to TTMA (a cationic gold nanoparticle coupled to 3,6,9,12-tetraoxatricosan-1-aminium, 23-mercapto-N,N,N-trimethyl) was studied by high-resolution turbidimetry (to observe a critical pH for binding), dynamic light scattering (to monitor particle growth), and isothermal titration calorimetry (to measure binding energetics), all as a function of pH and ionic strength. Distinctively higher affinities observed for BLG versus BSA, despite the lower pI of the latter, were explained in terms of their different charge anisotropies, namely, the negative charge patch of BLG. To confirm this effect, we studied two isoforms of BLG that differ in only two amino acids. Significantly stronger binding to BLGA could be attributed to the presence of the additional aspartates in the negative charge domain for the BLG dimer, best portrayed in DelPhi. This selectivity decreases at low ionic strength, at which both isoforms bind well below pI. Selectivity increases with ionic strength for BLG versus BSA, which binds above pI. This result points to the diminished role of long-range repulsions for binding above pI. Dynamic light scattering reveals a tendency for higher-order aggregation for TTMA-BSA at pH above the pI of BSA, due to its ability to bridge nanoparticles. In contrast, soluble BLG-TTMA complexes were stable over a range of pH because the charge anisotropy of this protein at makes it unable to bridge nanoparticles. Finally, isothermal titration calorimetry shows endoenthalpic binding for all proteins: the higher affinity of TTMA for BLGA versus BLGB comes from a difference in the dominant entropy term.

Protein purification by polyelectrolyte coacervation: influence of protein charge anisotropy on selectivity.

The effect of polyelectrolyte binding affinity on selective coacervation of proteins with the cationic polyelectrolyte, poly(diallyldimethylammonium chloride) (PDADMAC), was investigated for bovine serum albumin/ß-lactoglobulin (BSA/BLG) and for the isoforms BLG-A/BLG-B. High-sensitivity turbidimetric titrations were used to define conditions of complex formation and coacervation (pH(c) and pH(ϕ), respectively) as a function of ionic strength. The resultant phase boundaries, essential for the choice of conditions for selective coacervation for the chosen protein pairs, are nonmonotonic with respect to ionic strength, for both pH(c) and pH(ϕ). These results are explained in the context of short-range attraction/long-range repulsion governing initial protein binding "on the wrong side of pI" and also subsequent phase separation due to charge neutralization. The stronger binding of BLG despite its higher isoelectric point, inferred from lower pH(c), is shown to result from the negative "charge patch" on BLG, absent for BSA, as visualized via computer modeling (DelPhi). The higher affinity of BLG versus BSA was also confirmed by isothermal titration calorimetry (ITC). The relative values of pH(ϕ) for the two proteins show complex salt dependence so that the choice of ionic strength determines the order of coacervation, whereas the choice of pH controls the yield of the target protein. Coacervation at I = 100 mM, pH 7, of BLG from a 1:1 (w/w) mixture with BSA was shown by SEC to provide 90% purity of BLG with a 20-fold increase in concentration. Ultrafiltration was shown to remove effectively the polymer from the target protein. The relationship between protein charge anisotropy and binding affinity and between binding affinity and selective coacervation, inferred from the results for BLG/BSA, was tested using the isoforms of BLG. Substitution of glycine in BLG-B by aspartate in BLG-A lowers pH(c) by 0.2, as anticipated on the basis of DelPhi modeling. The stronger binding of BLG-A, confirmed by ITC, led to a difference in pH(ϕ) that was sufficient to provide enrichment by a factor of 2 for BLG-A in the coacervate formed from "native BLG".

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.

Advancing biocatalysis through enzyme, cellular, and platform engineering.

Biocatalysis offers opportunities for highly selective chemical reactions with high turnover rates under relatively mild conditions. Use of whole-cell or multi-enzyme systems enables transformations of complexity unmatched by nonbiological routes. However, advantages of biocatalysis are frequently compromised by poor enzymatic performance under non-native reaction conditions, the absence of enzymes with desired substrate or reaction specificities, and low metabolic fluxes or competing pathways. During the 234th National Meeting of the American Chemical Society, these issues were addressed in the "Advances in Biocatalysis" sessions. Protein engineering and metabolic pathway engineering were used to develop efficient enzymes and whole-cell catalysts. Novel strategies for the use of enzymes at solid interfaces and in nonaqueous environments were discussed, and efficient biotransformation platforms were demonstrated. These advances broaden the applications of biocatalysis in biofuels, pharmaceuticals, fine chemicals, and human health.

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.