Chunbo Lou

Genetic programs constructed from layered logic gates in single cells

Genetic programs function to integrate environmental sensors, implement signal processing algorithms and control expression dynamics. These programs consist of integrated genetic circuits that individually implement operations ranging from digital logic to dynamic circuits, and they have been used in various cellular engineering applications, including the implementation of process control in metabolic networks and the coordination of spatial differentiation in artificial tissues. A key limitation is that the circuits are based on biochemical interactions occurring in the confined volume of the cell, so the size of programs has been limited to a few circuits. Here we apply part mining and directed evolution to build a set of transcriptional AND gates in Escherichia coli. Each AND gate integrates two promoter inputs and controls one promoter output. This allows the gates to be layered by having the output promoter of an upstream circuit serve as the input promoter for a downstream circuit. Each gate consists of a transcription factor that requires a second chaperone protein to activate the output promoter. Multiple activator–chaperone pairs are identified from type III secretion pathways in different strains of bacteria. Directed evolution is applied to increase the dynamic range and orthogonality of the circuits. These gates are connected in different permutations to form programs, the largest of which is a 4-input AND gate that consists of 3 circuits that integrate 4 inducible systems, thus requiring 11 regulatory proteins. Measuring the performance of individual gates is sufficient to capture the behaviour of the complete program. Errors in the output due to delays (faults), a common problem for layered circuits, are not observed. This work demonstrates the successful layering of orthogonal logic gates, a design strategy that could enable the construction of large, integrated circuits in single cells.

Exploiting a precise design of universal synthetic modular regulatory elements to unlock the microbial natural products in Streptomyces

Significance To meet the increasing demands of drug discovery and biosynthetic studies, we established a precise quantitative method based on flow cytometry at single-cell (protoplast) resolution in Streptomyces for the identification of regulatory elements. A series of native or synthetic promoters and ribosomal binding sites has been characterized. Moreover, an insulator was demonstrated to eliminate element–element interference. As a proof of concept, a native silent gene cluster was activated by the synthetic modular regulatory elements in a predictable manner. The universality of these elements is of high value to the synthetic biology of Streptomyces. There is a great demand for precisely quantitating the expression of genes of interest in synthetic and systems biotechnology as new and fascinating insights into the genetics of streptomycetes have come to light. Here, we developed, for the first time to our knowledge, a quantitative method based on flow cytometry and a superfolder green fluorescent protein (sfGFP) at single-cell resolution in Streptomyces. Single cells of filamentous bacteria were obtained by releasing the protoplasts from the mycelium, and the dead cells could be distinguished from the viable ones by propidium iodide (PI) staining. With this sophisticated quantitative method, some 200 native or synthetic promoters and 200 ribosomal binding sites (RBSs) were characterized in a high-throughput format. Furthermore, an insulator (RiboJ) was recruited to eliminate the interference between promoters and RBSs and improve the modularity of regulatory elements. Seven synthetic promoters with gradient strength were successfully applied in a proof-of-principle approach to activate and overproduce the cryptic lycopene in a predictable manner in Streptomyces avermitilis. Our work therefore presents a quantitative strategy and universal synthetic modular regulatory elements, which will facilitate the functional optimization of gene clusters and the drug discovery process in Streptomyces.

Cas9-Assisted Targeting of CHromosome segments CATCH enables one-step targeted cloning of large gene clusters

The cloning of long DNA segments, especially those containing large gene clusters, is of particular importance to synthetic and chemical biology efforts for engineering organisms. While cloning has been a defining tool in molecular biology, the cloning of long genome segments has been challenging. Here we describe a technique that allows the targeted cloning of near-arbitrary, long bacterial genomic sequences of up to 100 kb to be accomplished in a single step. The target genome segment is excised from bacterial chromosomes in vitro by the RNA-guided Cas9 nuclease at two designated loci, and ligated to the cloning vector by Gibson assembly. This technique can be an effective molecular tool for the targeted cloning of large gene clusters that are often expensive to synthesize by gene synthesis or difficult to obtain directly by traditional PCR and restriction-enzyme-based methods.

A molecular model for persister in E. coli.

Like many other bacteria, Escherichia coli remain as tiny viable individuals named persisters after being exposed to an antibiotic. These persisters are believed to be phenotypic heterogeneous one rather than mutants, because their progenies are as susceptible to antibiotics as their ancestors. Recently, two persister-related genes (hipB/hipA) were confirmed to belong to a toxin-antitoxin (TA) module. Their control circuit was believed to be responsible for generation of the persister subpopulation. For the well-studied TA module, we build a simple genetic regulation model to explain the phenotypic heterogeneity. We find that a sole double-negative feedback loop is not enough to explain the phenotypic heterogeneity; the cooperation mechanisms in HipB and HipA are indispensable. Moreover, our model illustrates an important persister-related experimental phenomenon: the emergence of the persister depends on the growth rate in continuous culture.

Insulated transcriptional elements enable precise design of genetic circuits

Rational engineering of biological systems is often complicated by the complex but unwanted interactions between cellular components at multiple levels. Here we address this issue at the level of prokaryotic transcription by insulating minimal promoters and operators to prevent their interaction and enable the biophysical modeling of synthetic transcription without free parameters. This approach allows genetic circuit design with extraordinary precision and diversity, and consequently simplifies the design-build-test-learn cycle of circuit engineering to a mix-and-match workflow. As a demonstration, combinatorial promoters encoding NOT-gate functions were designed from scratch with mean errors of <1.5-fold and a success rate of >96% using our insulated transcription elements. Furthermore, four-node transcriptional networks with incoherent feed-forward loops that execute stripe-forming functions were obtained without any trial-and-error work. This insulation-based engineering strategy improves the resolution of genetic circuit technology and provides a simple approach for designing genetic circuits for systems and synthetic biology.Unwanted interactions between cellular components can complicate rational engineering of biological systems. Here the authors design insulated minimal promoters and operators that enable biophysical modeling of bacterial transcription without free parameters for precise circuit design.

Paired Design of dCas9 as a Systematic Platform for the Detection of Featured Nucleic Acid Sequences in Pathogenic Strains

We developed an in vitro DNA detection system using a pair of dCas9 proteins linked to split halves of luciferase. Luminescence was induced upon colocalization of the reporter pair to a ∼44 bp target sequence defined by sgRNAs. We used the system to detect Mycobacterium tuberculosis DNA with high specificity and sensitivity. The reprogrammability of dCas9 was further leveraged in an array design that accesses sequence information across the entire genome.

Measurements of Gene Expression at Steady State Improve the Predictability of Part Assembly.

Mathematical modeling of genetic circuits generally assumes that gene expression is at steady state when measurements are performed. However, conventional methods of measurement do not necessarily guarantee that this assumption is satisfied. In this study, we reveal a bi-plateau mode of gene expression at the single-cell level in bacterial batch cultures. The first plateau is dynamically active, where gene expression is at steady state; the second plateau, however, is dynamically inactive. We further demonstrate that the predictability of assembled genetic circuits in the first plateau (steady state) is much higher than that in the second plateau where conventional measurements are often performed. By taking the nature of steady state into consideration, our method of measurement promises to directly capture the intrinsic property of biological parts/circuits regardless of circuit-host or circuit-environment interactions.

d-Sedoheptulose-7-phosphate is a common precursor for the heptoses of septacidin and hygromycin B

Significance Septacidin and its analogs are potential anticancer and pain-relief drugs. Hygromycin B is an anthelmintic agent practically used in swine and poultry farming. A common feature of these compounds is that they all have heptose moieties. Here we show that the heptoses of septacidin and hygromycin B are both derived from d-sedoheptulose-7-phosphate but are biosynthesized through different pathways. Septacidin producer, a gram-positive bacterium, shares the same ADP-heptose biosynthesis pathway with gram-negative bacterium lipopolysaccharide biosynthesis. These findings not only elucidate the biosynthesis mechanisms of septacidin and hygromycin B but enable opportunities for manipulation of their heptose moieties by combinatorial biosynthesis and for changing the structure of heptoses in gram-negative bacterium lipopolysaccharides. Seven-carbon-chain–containing sugars exist in several groups of important bacterial natural products. Septacidin represents a group of l-heptopyranoses containing nucleoside antibiotics with antitumor, antifungal, and pain-relief activities. Hygromycin B, an aminoglycoside anthelmintic agent used in swine and poultry farming, represents a group of d-heptopyranoses–containing antibiotics. To date, very little is known about the biosynthesis of these compounds. Here we sequenced the genome of the septacidin producer and identified the septacidin gene cluster by heterologous expression. After determining the boundaries of the septacidin gene cluster, we studied septacidin biosynthesis by in vivo and in vitro experiments and discovered that SepB, SepL, and SepC can convert d-sedoheptulose-7-phosphate (S-7-P) to ADP-l-glycero-β-d-manno-heptose, exemplifying the involvement of ADP-sugar in microbial natural product biosynthesis. Interestingly, septacidin, a secondary metabolite from a gram-positive bacterium, shares the same ADP-heptose biosynthesis pathway with the gram-negative bacterium LPS. In addition, two acyltransferase-encoding genes sepD and sepH, were proposed to be involved in septacidin side-chain formation according to the intermediates accumulated in their mutants. In hygromycin B biosynthesis, an isomerase HygP can recognize S-7-P and convert it to ADP-d-glycero-β-d-altro-heptose together with GmhA and HldE, two enzymes from the Escherichia coli LPS heptose biosynthetic pathway, suggesting that the d-heptopyranose moiety of hygromycin B is also derived from S-7-P. Unlike the other S-7-P isomerases, HygP catalyzes consecutive isomerizations and controls the stereochemistry of both C2 and C3 positions.

Rational Design of an Ultrasensitive Quorum-Sensing Switch

One of the purposes of synthetic biology is to develop rational methods that accelerate the design of genetic circuits, saving time and effort spent on experiments and providing reliably predictable circuit performance. We applied a reverse engineering approach to design an ultrasensitive transcriptional quorum-sensing switch. We want to explore how systems biology can guide synthetic biology in the choice of specific DNA sequences and their regulatory relations to achieve a targeted function. The workflow comprises network enumeration that achieves the target function robustly, experimental restriction of the obtained candidate networks, global parameter optimization via mathematical analysis, selection and engineering of parts based on these calculations, and finally, circuit construction based on the principles of standardization and modularization. The performance of realized quorum-sensing switches was in good qualitative agreement with the computational predictions. This study provides practical principles for the rational design of genetic circuits with targeted functions.

Engineering of a genetic circuit with regulatable multistability

Synthetic biologists are dedicated to designing genetic systems from the bottom up to understand how living systems work. To date, a variety of genetic circuits exhibiting bistability have been designed, greatly expanding our understanding of the biological multistability in natural systems. However, the study of more complex forms of biological multistability using synthetic methods is still limited. In this report, we describe the engineering of a genetic circuit with regulatable multistability. A novel genetic toggle switch exhibiting inducible bistability and a self-activation circuit were individually designed and characterized, after which they were assembled to create a circuit that presents tristability. In bacteria, this synthetic circuit enables cells to differentiate spontaneously into three different states of gene expression. Moreover, the multistability of the circuit can be modulated by external inputs. This work provides a synthetic biology framework for the study of biological multistability and may help to understand natural multistability phenomena.