ACS Synthetic Biology

The fast-growing microbe Vibrio natriegens is capable of natural transformation where it draws DNA in from media via an active process under physiological conditions. Using an engineered strain with a genomic copy of the master competence regulator tfoX from Vibrio cholera in combination with a new minimal competence media (MCM) that uses acetate as an energy source, we demonstrate naturally competent cells which are created, transformed, and recovered entirely in the same media, without exchange or addition of new media. Cells are naturally competent to plasmids, recombination with linear DNA, and co-transformation of both to select for scarless and markerless genomic edits. The entire process is simple and inexpensive, requiring no capital equipment for an entirely room temperature process (Zero Capital protocol, 104 cfu/{micro}g), or just an incubator (High Efficiency protocol, 105-6 cfu/{micro}g). These cells retain their naturally competent state when frozen and are transformable immediately upon thawing like a typical chemical or electrochemical competent cell. Since the optimized transformation protocol requires only 50 minutes of hands-on time, and V. natriegens grows quickly even on plates, a transformation started at 9 AM yields abundant culturable single colonies by 5 PM. Further, because all stages of transformation occur in the same media, and the process can be arbitrarily scaled in volume, this natural competence strain and media could be ideal for automated directed evolution applications. As a result, naturally competent V. natriegens could compete with E. coli as an excellent chassis for low-cost and highly scalable synthetic biology.

1Bi-stable gene regulatory motifs are found in a wide variety of natural gene regulatory networks and effect transcriptional switching between stable phenotypic states in cells. In synthetic gene regulatory circuits, these architectures can be leveraged to dynamically switch between distinct metabolic states for metabolic engineering and therapeutic applications. However, the lack of modularity and predictability of these motifs in varying environments has limited widespread application, especially since the factors that affect switching characteristics are still unclear. In this work, we use a mathematical model along with a newly developed dynamical modeling and continuity analysis framework to analyze the dynamics and robustness of bi-stable switches over a range of biologically relevant model parameter values. We identify a hitherto undiscovered fundamental trade-off between the robustness of the motif - the parameter ranges over which it retains bi-stable function, and the speed at which it effects a phenotypic change. Further, using E. coli as a model host, we constructed a large library (100) of transcriptional switches that show a wide range of switching speeds, to experimentally demonstrate the presence of this trade-off. The presence of this trade-off has significant implications on the design of transcriptional switches for diverse applications and explains the circuit architecture of naturally occurring transcriptional switches as well. Additionally, we anticipate that our diverse library of experimentally validated robust bi-stable switches will be valuable to effect phenotypic changes with differing switching speed requirements for metabolic engineering applications. 2 Significance statementThe ability to switch genes on and off in response to spatio-temporal stimuli is critical to the survival of all organisms. At the cellular level, such switching is effected by regulatory motifs such as the bi-stable genetic toggle switch. Bi-stable motifs also serve as a primitive mode of cellular memory - "remembering" the last environment to which the cells have been exposed. In this work, we uncover a fundamental trade-off between the robust functioning of these switches and the speed at which they can effect a change in the gene expression landscape. These findings have a broad impact on the design and use of such synthetic gene regulatory devices across several fields such as industrial biotechnology, healthcare, etc.

Synthetic biology is as an excellent vehicle for education, as it enables creative combination of engineering and molecular biology approaches for quantitative characterisations of the assembled constructs. However, there is a limited number of resources available for such applications in the educational context, where straightforward setup, easily measurable phenotypes and extensibility are of particular importance. To expand the availability of education-friendly resources to teach synthetic biology and genetic engineering, we developed Unigems, a set of 10 plasmids that enable out-of-the-box investigations of principles of gene expression control, as well as more complex designs a biological logic gate. The system uses a common high-copy plasmid backbone and a common set of primers to enable Gibson-assembly of PCR-generated or synthesised parts into a target vector. It currently has two reporter genes with either two constitutive (high- or low-level) or two inducible (lactose- or arabinose-) promoters, as well as a single-plasmid implementation of an AND logic gate. The Unigems system has already been employed in undergraduate teaching settings, during outreach events and for training of iGEM teams. All plasmids have been deposited in Addgene.

Cell-free synthetic biology biosensors have potential as effective in vitro diagnostic technologies for the detection of chemical compounds such as toxins and human health biomarkers. They have several advantages over conventional laboratory-based diagnostic approaches, including being able to be assembled, freeze-dried, distributed, and then used at the point-of-need. This makes them an attractive platform for cheap and rapid chemical detection across the globe. Though promising, a major challenge is scaling up biosensor manufacturing to meet the needs of their multiple uses. Currently, cell-free biosensor assembly during lab-scale development is mostly performed manually by the operator, leading to quality control and performance variability issues. Here we explore the use of liquid handling robotics to manufacture cell-free biosensor reactions. We compare both manual and semi-automated reaction assembly approaches using the Opentrons OT-2 liquid handling platform on two different cell-free gene expression assay systems that constitutively produce colorimetric (LacZ) or fluorescent (GFP) signals. We test the designed protocol by constructing an entire 384-well plate of fluoride sensing cell-free biosensors and demonstrate that they perform closely to expected detection outcomes.

The discovery of new genes regulating essential biological processes has become increasingly important, and CRISPRi has emerged as a powerful tool for achieving this goal. This method has been used in many model organisms to decrease the expression of specific genes and assess their impact on phenotype. Pooled CRISPRi libraries in bacteria have been particularly useful in discovering new regulators of growth, division, and other biological processes. However, these libraries rely on the induction of dCas9 via an inducible promoter, which can be problematic due to promoter leakiness. This is a widespread phenomenon of any inducible promoter that can result in the unwanted downregulation of genes and the emergence of genetic suppressors when essential genes are knocked down. To overcome this issue, we have developed a novel strategy that eliminates dCas9 leakiness and enables reversible knockdown control using the rapamycin-dependent degron system in Bacillus subtilis. This degron system causes rapid degradation of dCas9, resulting in an almost instant reset of the system. Our results demonstrate that it is possible to achieve zero CRISPRi activity in the uninduced state and full activity in the induced state. This improved CRISPRi system will enable researchers to investigate phenotypic changes more effectively while reducing the undesirable effects of leaky expression and noise in their phenotypic data. Moreover, a rapid degradation system could serve as a tool for dynamic perturbation before compensation mechanisms or stress responses kick in. Finally, this approach can be adapted to other organisms and other promoter-inducible systems, potentially opening up strategies for tighter control of gene expression.

Microbial optogenetic tools can regulate gene expression with high spatial and temporal precision, offering excellent potential for single-cell resolution studies. However, bacterial optogenetic systems have primarily been deployed for population-level experiments. It is not always clear how these tools perform in single cells, where stochastic effects can be substantial. In this study, we focus on optogenetic Cre recombinase and systematically compare the performance of three variants (OptoCre-REDMAP, OptoCre-Vvd, and PA-Cre) for their population-level and single-cell activity. We quantify recombination efficiency, expression variability, and activation dynamics using reporters which produce changes in fluorescence or antibiotic resistance following light-induced Cre activity. Our results indicate that optogenetic recombinase performance can be reporter-dependent. Further, single-cell analysis revealed highly heterogeneous activity across cells. Although general trends match expectations for light-dependent recombination, we found substantial variation in the efficiency and timing of recombinase activity from cell to cell. These findings suggest critical criteria for selecting optogenetic recombinase systems and indicate areas for optimization to improve single-cell capabilities of bacterial optogenetic tools.

Here we report the development of a conditional degron system for use in bacteria in which a tagged fluorescent protein (FP) reporter, mVenus-S1, is stabilized only in the presence of TAR RNA. The functional design takes advantage of the bipartite nature of the ssrA degron sequence, so that TAR RNA binding to the inserted Tat peptide sequence blocks degron recognition to rescue protein activity. Several fluorescent proteins and a chemiluminescent enzyme can be tagged and activated using this system, and our results reveal that chromophore maturation time correlates with fold change, with up to [~]60-fold turn-on. Finally, we designed a functional biosensor for oxyS sRNA and integrated it into a single transcript in which the FP and RNA-based biosensor are encoded on the same mRNA. To our knowledge, this is the first proof-of-concept for a novel biosensor design that expresses both protein and RNA components from a single transcript. The streamlined system is fully genetically encoded, does not require an exogenous fluorophore, and permits both components to be coordinated by expression from the same promoter.

Control of the lac operon with IPTG has been used for decades to regulate gene expression in E. coli for countless applications, including metabolic engineering and recombinant protein production. However, optogenetics offers unique capabilities such as easy tunability, reversibility, dynamic induction strength, and spatial control that are difficult to obtain with chemical inducers. We developed an optogenetic lac operon in a series of circuits we call OptoLAC. With these circuits, we control gene expression from various IPTG-inducible promoters using only blue light. Applying them to metabolic engineering improves mevalonate and isobutanol production by 24% and 27% respectively, compared to IPTG induction, in light-controlled fermentations scalable to at least 2L bioreactors. Furthermore, OptoLAC circuits enable light control of recombinant protein production, reaching yields comparable to IPTG induction, but with enhanced tunability of expression and spatial control. OptoLAC circuits are potentially useful to confer light controls over other cell functions originally engineered to be IPTG-inducible.

CRISPR-mediated gene activation (CRISPRa) allows concurrent transcriptional activation of many genes and has found widespread use in genome-wide screening, bioproduction, and therapeutics. Scaffold RNAs (scRNAs) recruit dCas9 and an activator protein (RBP-AD) to the target gene for activation with high sequence specificity. Here, we show that, despite this specificity, orthogonal scRNAs interfere with each other because they compete for dCas9 and RBP-AD. Specifically, we demonstrate that the expression of an scRNA that binds to these resources results in repression of genes targeted by different scRNAs. Intriguingly, we also discover that transcriptional gene regulation by an scRNA is biphasic, wherein increased level of the scRNA leads to gene repression instead of activation. These effects are significant even when dCas9 and RBP-AD are expressed at the maximum level tolerable by the cell. Our results demonstrate that CRISPRa systems are not as modular as previously thought and establish predictive modeling tools to assess the emergent behavior of multi-module CRISPRa networks.

Lactic acid bacteria (LAB) are important for many biotechnological applications, such as bioproduction and engineered probiotics for therapy. Inducible promoters are key gene expression control elements, yet those available in LAB are mainly based on bacteriocin systems and have many drawbacks, including large gene clusters, costly inducer peptides and little portability to in vivo settings. Using Lactobacillus gasseri, a model commensal bacteria from the human gut, we report the engineering of Lactospanks promoters (Pls), a collection of variable strength inducible promoters controlled by the LacI repressor from B. subtilis and induced by isopropyl {beta}-D-1-thiogalactopyranoside (IPTG). We first show that the Phyper-spank promoter from Bacillus subtilis is functional in L. gasseri, albeit with substantial leakage. We then construct and screen a semi-rational library of Phyper-spank variants to select a set of four IPTG-inducible promoters that span a range of expression levels and exhibit reduced leakages and operational dynamic ranges (from ca. 9 to 28 fold-change). With their low genetic footprint and simplicity of use, Lactospanks will support many applications in L. gasseri, and potentially other lactic acid and gram-positive bacteria.

Engineered programmable RNA sensors have been applied in low-cost diagnostics, endogenous RNA detection, and multi-input genetic circuits. However, designing, producing, and screening high-performance RNA sensors remains time consuming and labor intensive. Here, we present an automated plasmid assembly pipeline using liquid handling robotics to enable high-throughput construction of plasmids with arbitrary sequences. We compare automated and manual assembly methods using the NGS Hamilton Microlab STAR across two plasmid backbones to evaluate efficiency and reliability. As a proof of concept, we use this modular platform to construct 144 total plasmids encoding riboregulators targeting diverse viral targets along with their cognate trigger sequences. We further demonstrate that the assembled plasmids are functional in both bacterial and cell-free expression systems.

The ability to modulate gene expression is crucial for studying gene function and programming cell behaviors. Combining the reliability of CRISPRi and the precision of optogenetics, the opto-CRISPRi technique is emerging as an advanced tool for live-cell gene regulation. Since previous versions of opto-CRISPRi often exhibit a no more than 10 folds dynamic range due to the leakage activity, they are not suitable for targets that are sensitive to such leakage or critical for cell growth. Here, we describe a green-light activated CRISPRi system with high dynamic range (40-fold) and the flexibility of changing targets in Escherichia coli. Our optoCRISPRi-HD system can efficiently repress essential genes, non-essential genes or inhibit the initiation of DNA replication. Providing a regulative system with high resolution over space-time and extensive targets, our study would facilitate further researches involving complex gene networks, metabolic flux redirection or bioprinting.

Recently, a new subset of fluorescent proteins has been identified from the Aequorea species of jellyfish. These fluorescent proteins were characterized in vivo; however, there has not been validation of these proteins within cell-free systems. Cell-free systems and technology development is a rapidly expanding field, encompassing foundational research, synthetic cells, bioengineering, biomanufacturing and drug development. Cell-free systems rely heavily on fluorescent proteins as reporters. Here we characterize and validate this new set of Aequorea proteins for use in a variety of cell-free and synthetic cell expression platforms. O_FIG O_LINKSMALLFIG WIDTH=144 HEIGHT=200 SRC="FIGDIR/small/519681v1_ufig1.gif" ALT="Figure 1"> View larger version (41K): org.highwire.dtl.DTLVardef@3a3100org.highwire.dtl.DTLVardef@672233org.highwire.dtl.DTLVardef@f65f32org.highwire.dtl.DTLVardef@e40aaf_HPS_FORMAT_FIGEXP M_FIG C_FIG

Photosensory protein domains are the basis of optogenetic protein engineering. These domains originate from natural sources where they fulfill specific functions ranging from the protection against photooxidative damage to circadian rhythms. When used in synthetic biology, the features of these photosensory domains can be specifically tailored towards the application of interest, enabling their full exploitation for optogenetic regulation in basic research and applied bioengineering. In this work, we develop and apply a simple, yet powerful, directed evolution and high-throughput screening strategy that allows us to alter the most fundamental property of the widely used nMag/pMag photodimerization system: its light sensitivity. We identify a set of mutations located within the photosensory domains, which either increase or decrease the light sensitivity at sub-saturating light intensities, while also improving the dark-to-light fold change in certain variants. For some of these variants, photosensitivity and expression levels could be changed independently, showing that the shape of the light-activity dose-response curve can be tuned and adjusted. We functionally characterize the variants in vivo in bacteria on the single-cell and the population levels. We further show that a subset of these variants can be transferred into the mOptoT7 for gene expression regulation in mammalian cells. We demonstrate increased gene expression levels for low light intensities, resulting in reduced potential phototoxicity in long-term experiments. Our findings expand the applicability of the widely used Magnets photosensors by enabling a tuning towards the needs of specific optogenetic regulation strategies. More generally, our approach will aid optogenetic approaches by making the adaptation of photosensor properties possible to better suit specific experimental or bioprocess needs.

A universal biochemical signal for bacterial cell-cell communication could facilitate programming dynamic responses in diverse bacterial consortia. However, the classical quorum sensing paradigm is that gram-negative and gram-positive bacteria generally communicate via homoserine lactones (HSL) or oligopeptide molecular signals, respectively, to elicit population responses. Here, we create synthetic HSL sensors for gram-positive Bacillus subtilis 168 using allosteric LuxR-type regulators (RpaR, LuxR, RhlR, and CinR) and synthetic promoters. Promoters were combinatorially designed from different sequence elements (-35, -16, -10, and transcriptional start regions). We quantified the effects of these combinatorial promoters on sensor activity and determined how regulator expression affects its activation, achieving up to 293-fold activation. Using statistical design of experiments, we identified significant effects of promoter regions and pairwise interactions on sensor activity, which helped to understand the sequence-function relationships for synthetic promoter design. We present the first known set of functional HSL sensors ([≥] 20-fold dynamic range) in B. subtilis for four different HSL chemical signals: p-coumaroyl-HSL, 3-oxohexanoyl-HSL, n-butyryl-HSL, and n-(3-hydroxytetradecanoyl)-HSL. This set of synthetic HSL sensors for a gram-positive bacterium can pave the way for designable interspecies communication within microbial consortia.

The soil dwelling bacteria Streptomyces is an abundant producer of numerous anticancer, antifungal, and antibiotic compounds (i.e. Natural Products, NPs). The sophisticated cellular machinery required to produce NPs is frequently regulated by quorum-sensing systems, consisting of cluster situated regulators (CSRs), such as TetR-like repressors, and small-molecule autoregulator (AR) ligands. Only a small fraction of bioinformatically predicted quorum-sensing AR circuits have been experimentally determined, and fewer still have been engineered as inducible expression systems for synthetic biology. This research details the development of eight CSR-based AR biosensors and the synthetic routes to their AR ligands. Overall, the AR biosensors exhibit a range of maximum activation, AR affinity, and AR selectivity. We examined crosstalk between noncognate CSRs and ARs, as well as the ability of CSRs to regulate alternative operators. Additionally, we establish these biosensors can be cocultured with Streptomyces for rapid analysis of AR production. Finally, we demonstrate the CSR-based biosensor vectors can be combined to create orthogonal signaling systems in bacterial coculturing or multi-input genetic circuits. Longterm, these Streptomyces AR biosensors will contribute to the elucidation of small molecule quorum sensing circuits employed by Streptomyces as well as increasing the complexity of genetic circuits used in industrial or agricultural settings.

Genetic systems engineering is constrained by high DNA synthesis costs, assembly inefficiencies, and challenges in expressing complex proteins. To address these limitations, we developed a highly parallel, low-cost pipeline for the design, assembly, and functional screening of genetic systems, which we stress-tested on highly repetitive structural proteins, including spider silk, biocements, reflectins, and talins. The integrated pipeline combines computational genetic systems design, low-cost many-plasmid DNA assembly from oligopools, automated many-to-many mapping using nanopore sequencing data, and a label-free biosensor to measure single-cell protein expression levels. We applied this pipeline to build 240 plasmids, achieving an 88% success rate (up to 2000 bp) using standard clonal isolation and 58% assembly efficiency (up to 5600 bp) without selective DNA purification, while lowering material costs by up to 24-fold. We applied the biosensor to identify genetic factors that create distinct cellular subpopulations with varying protein expression levels. Overall, the integrated pipeline will dramatically lower the cost of high-throughput synthetic biology, while demonstrating how designing genetic systems to improve build efficiency ("design for build") and directly incorporating biosensors into genetic systems ("design for test") will greatly accelerate design-build-test workflows.

Cell-free transcription-translation (TXTL) systems expressing genes from linear dsDNA enable rapid prototyping of genetic devices while avoiding cloning steps. However, repetitive inclusion of a reporter gene is an incompressible cost and sometimes accounts for most of the synthesized DNA length. Here we present minireporters based on split-GFP systems that reassemble into functional fluorescent proteins and can be used to monitor gene expression in E. coli TXTL. The 135 bp GFP10-11 fragment produces a fluorescent signal comparable to its full-length GFP counterpart when reassembling with its complementary protein synthesized from the 535 bp fragment expressed in TXTL. We show that minireporters can be used to characterize promoter libraries, with data qualitatively comparable to full-length GFP, and matching with in vivo expression measurements. We also use minireporters as small fusion tags to measure TXTL protein and peptide production yield. Finally, we generalize our concept by providing a luminescent minireporter based on split-nanoluciferase. The [~]80% gene sequence length reduction afforded by minireporters lowers synthesis costs and liberates space for testing larger devices while producing a reliable output. In the peptide production context, the small size of minireporters compared to full-length GFP is less likely to bias peptide solubility assays. We anticipate that minireporters will facilitate rapid and cost-efficient genetic device prototyping, protein production, and interaction assays.

Context independent gene expression is required for genetic circuits to maintain consistent and predicable behavior. Previous efforts to develop context independent translation have leveraged the helicase activity of translating ribosomes via bicistronic design translational control elements (BCDs) located within an efficiently translated leader peptide. We have developed a series of bicistronic translational control elements with strengths that span several orders of magnitude, maintain consistent expression levels across diverse sequence contexts, and are agnostic to common ligation sequences used in modular cloning systems. We have used this series of BCDs to investigate several features of this design, including the spacing of the start and stop codons, the nucleotide identity upstream of the start codon, and factors affecting translation of the leader peptide. To demonstrate the flexibility of this architecture and their value as a generic modular expression control cassette for synthetic biology, we have developed a set of robust BCDs for use in several Rhodococcus species.

Whole-cell biosensors are emerging as promising tools for monitoring environmental pollutants such as heavy metals. These sensors constitute a genetic circuit comprising a sensing module and an output module, such that a detectable signal is produced in the presence of the desired analyte. The MerR family of metal-responsive regulators offers great potential for the construction of metal sensing circuits, due to their high sensitivity, tight transcription control and large diversity in metal-specificity. However, the sensing diversity is broadest in Gram-negative systems, while chassis organisms are often selected from Gram-positive species, particularly sporulating bacilli. This can be problematic, because Gram-negative biological parts, such as promoters, are frequently observed to be non-functional in Gram-positive hosts. Herein, we combined construction of synthetic genetic circuits and chimeric MerR regulators, supported by structure-guided design, to generate metal-sensitive biosensor modules that are functional in the biotechnological work-horse species Bacillus subtilis. These chimeras consist of a constant Gram-positive derived DNA-binding domain fused to variable metal binding domains of Gram-negative origins. To improve the specificity of the whole-cell biosensor, we developed a modular AND gate logic system based on the B. subtilis natively split {sigma}-factor, SigO-RsoA, designed to maximise future use for synthetic biology applications in B. subtilis. This work provides insights into the use of modular regulators, such as the MerR family, in the design of synthetic circuits for the detection of heavy metals, with potential wider applicability of the approach to other systems and genetic backgrounds.