Once an engineered organism completes its task, it is useful to degrade the associated DNA to reduce environmental release and protect intellectual property. Here we present a genetically encoded device (DNAi) that responds to a transcriptional input and degrades user-defined DNA. This enables engineered regions to be obscured when the cell enters a new environment. DNAi is based on type-IE CRISPR biochemistry and a synthetic CRISPR array defines the DNA target(s). When the input is on, plasmid DNA is degraded 108-fold. When the genome is targeted, this causes cell death, reducing viable cells by a factor of 108. Further, the CRISPR nuclease can direct degradation to specific genomic regions (for example, engineered or inserted DNA), which could be used to complicate recovery and sequencing efforts. DNAi can be stably carried in an engineered organism, with no impact on cell growth, plasmid stability or DNAi inducibility even after passaging for >2 months.
The ability to contain and destroy synthetically engineered microorganisms is an important consideration with environmental, industrial and intellectual property implications. Here Caliando et al. design and demonstrate a stably integrated CRISPR-based system for targeted DNA destruction.
Genetic memory enables the recording of information in the DNA of living cells. Memory can record a transient environmental signal or cell state that is then recalled at a later time. Permanent memory is implemented using irreversible recombinases that invert the orientation of a unit of DNA, corresponding to the [0,1] state of a bit. To expand the memory capacity, we have applied bioinformatics to identify 34 phage integrases (and their cognate attB and attP recognition sites), from which we build 11 memory switches that are perfectly orthogonal to each other and the FimE and HbiF bacterial invertases. Using these switches, a memory array is constructed in Escherichia coli that can record 1.375 bytes of information. It is demonstrated that the recombinases can be layered and used to permanently record the transient state of a transcriptional logic gate.
Synthetic biology; systems biology; biotechnology; genetic circuit; part mining
Genetic circuits require many regulatory parts in order to implement signal processing or execute
algorithms in cells. A potentially scalable approach is to use dCas9, which employs small guide RNAs
(sgRNAs) to repress genetic loci via the programmability of RNA:DNA base pairing. To this end, we
use dCas9 and designed sgRNAs to build transcriptional logic gates and connect them to perform
computation in living cells. We constructed a set of NOT gates by designing five synthetic
Escherichia coli σ70 promoters that are repressed by
corresponding sgRNAs, and these interactions do not exhibit crosstalk between each other. These
sgRNAs exhibit high on-target repression (56- to 440-fold) and negligible off-target interactions
(< 1.3-fold). These gates were connected to build larger circuits, including the
Boolean-complete NOR gate and a 3-gate circuit consisting of four layered sgRNAs. The synthetic
circuits were connected to the native E. coli regulatory network by designing
output sgRNAs to target an E. coli transcription factor (malT).
This converts the output of a synthetic circuit to a switch in cellular phenotype (sugar
utilization, chemotaxis, phage resistance).
CRISPR; genetic compiler; synthetic biology; TALE; TetR homologue
Cells are able to navigate environments, communicate, and build complex patterns by initiating gene expression in response to specific signals. Engineers need to harness this capability to program cells to perform tasks or build chemicals and materials that match the complexity seen in nature. This review describes new tools that aid the construction of genetic circuits. We show how circuit dynamics can be influenced by the choice of regulators and changed with expression “tuning knobs.” We collate the failure modes encountered when assembling circuits, quantify their impact on performance, and review mitigation efforts. Finally, we discuss the constraints that arise from operating within a living cell. Collectively, better tools, well-characterized parts, and a comprehensive understanding of how to compose circuits are leading to a breakthrough in the ability to program living cells for advanced applications, from living therapeutics to the atomic manufacturing of functional materials.
Methylating chemicals are common in industry and agriculture and are often toxic, partly due to their propensity to methylate DNA. The Escherichia coli Ada protein detects methylating compounds by sensing aberrant methyl adducts on the phosphoester backbone of DNA. We characterize this system as a genetic sensor and engineer it to lower the detection threshold. By overexpressing Ada from a plasmid, we improve the sensor’s dynamic range to 350-fold induction and lower its detection threshold to 40 µM for methyl iodide. In eukaryotes, there is no known sensor of methyl adducts on the phosphoester backbone of DNA. By fusing the N-terminal domain of Ada to the Gal4 transcriptional activation domain, we built a functional sensor for methyl phosphotriester adducts in Saccharomyces cerevisiae. This sensor can be tuned to variable specifications by altering the expression level of the chimeric sensor and changing the number of Ada operators upstream of the Gal4-sensitive reporter promoter. These changes result in a detection threshold of 28 µM and 5.2-fold induction in response to methyl iodide. When the yeast sensor is exposed to different SN1 and SN2 alkylating compounds, its response profile is similar to that observed for the native Ada protein in E. coli, indicating that its native function is retained in yeast. Finally, we demonstrate that the specifications achieved for the yeast sensor are suitable for detecting methylating compounds at relevant concentrations in environmental samples. This work demonstrates the movement of a sensor from a prokaryotic to eukaryotic system and its rational tuning to achieve desired specifications.
synthetic biology; genetic circuit; genetic device; fumigant; Gal4
Genetic circuits perform computational operations based on interactions between freely diffusing molecules within a cell. When transcription factors are combined to build a circuit, unintended interactions can disrupt its function. Here, we apply “part mining” to build a library of 73 TetR-family repressors gleaned from prokaryotic genomes. The operators of a subset were determined using an in vitro method and this information was used to build synthetic promoters. The promoters and repressors were screened for cross-reactions. Of these, 16 were identified that both strongly repress their cognate promoter (5- to 207-fold) and do not interact with other promoters. Each repressor:promoter pair was converted to a NOT gate and characterized. Used as a set of 16 NOR gates, there are >1054 circuits that could be built by changing the pattern of input and output promoters. This represents a large set of compatible gates that can be used to construct user-defined circuits.
Synthetic genetic systems share resources with the host, including machinery for transcription
and translation. Phage RNA polymerases (RNAPs) decouple transcription from the host and generate
high expression. However, they can exhibit toxicity and lack accessory proteins (σ factors
and activators) that enable switching between different promoters and modulation of activity. Here,
we show that T7 RNAP (883 amino acids) can be divided into four fragments that have to be
co-expressed to function. The DNA-binding loop is encoded in a C-terminal 285-aa ‘σ
fragment’, and fragments with different specificity can direct the remaining 601-aa
‘core fragment’ to different promoters. Using these parts, we have built a resource
allocator that sets the core fragment concentration, which is then shared by multiple σ
fragments. Adjusting the concentration of the core fragment sets the maximum transcriptional
capacity available to a synthetic system. Further, positive and negative regulation is implemented
using a 67-aa N-terminal ‘α fragment’ and a null (inactivated) σ
fragment, respectively. The α fragment can be fused to recombinant proteins to make promoters
responsive to their levels. These parts provide a toolbox to allocate transcriptional resources via
different schemes, which we demonstrate by building a system which adjusts promoter activity to
compensate for the difference in copy number of two plasmids.
genetic circuit; resource allocation; split protein; synthetic biology; T7 RNA polymerase
Synthetic genetic programs are built from circuits that integrate sensors and implement temporal control of gene expression1–4. Transcriptional circuits are layered by using promoters to carry the signal between circuits. In other words, the output promoter of one circuit serves as the input promoter to the next. Thus, connecting circuits requires physically connecting a promoter to the next circuit. We show that the sequence at the junction between the input promoter and circuit can affect the input-output response (transfer function) of the circuit5–9. A library of putative sequences that might reduce (or buffer) such context effects, which we refer to as ‘insulator parts’, is screened in Escherichia coli. We find that ribozymes that cleave the 5′ untranslated region (5′-UTR) of the mRNA are effective insulators. They generate quantitatively identical transfer functions, irrespective of the identity of the input promoter. When these insulators are used to join synthetic gene circuits, the behavior of layered circuits can be predicted using a mathematical model. The inclusion of insulators will be critical in reliably permuting circuits to build different programs.
Genetic programs function to integrate environmental sensors, implement signal processing algorithms and control expression dynamics1. These programs consist of integrated genetic circuits that individually implement operations ranging from digital logic to dynamic circuits2–6, 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 circuits1,7. 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.
Computation underlies the organization of cells into higher-order structures, for example during development or the spatial association of bacteria in a biofilm1–3. Each cell performs a simple computational operation, but when combined with cell–cell communication, intricate patterns emerge. Here we study this process by combining a simple genetic circuit with quorum sensing to produce more complex computations in space. We construct a simple NOR logic gate in Escherichia coli by arranging two tandem promoters that function as inputs to drive the transcription of a repressor. The repressor inactivates a promoter that serves as the output. Individual colonies of E. coli carry the same NOR gate, but the inputs and outputs are wired to different orthogonal quorum-sensing ‘sender’ and ‘receiver’ devices4,5. The quorum molecules form the wires between gates. By arranging the colonies in different spatial configurations, all possible two-input gates are produced, including the difficult XOR and EQUALS functions. The response is strong and robust, with 5- to >300-fold changes between the ‘on’ and ‘off’ states. This work helps elucidate the design rules by which simple logic can be harnessed to produce diverse and complex calculations by rewiring communication between cells.
Synthetic genetic programs promise to enable novel applications in industrial processes. For such applications, the genetic circuits that compose programs will require fidelity in varying and complex environments. In this work, we report the performance of two synthetic circuits in Escherichia coli under industrially relevant conditions, including the selection of media, strain, and growth rate. We test and compare two transcriptional circuits: an AND and a NOR gate. In E. coli DH10B, the AND gate is inactive in minimal media; activity can be rescued by supplementing the media and transferring the gate into the industrial strain E. coli DS68637 where normal function is observed in minimal media. In contrast, the NOR gate is robust to media composition and functions similarly in both strains. The AND gate is evaluated at three stages of early scale-up: 100 ml shake-flask experiments, a 1 ml MTP microreactor, and a 10 L bioreactor. A reference plasmid that constitutively produces a GFP reporter is used to make comparisons of circuit performance across conditions. The AND gate function is quantitatively different at each scale. The output deteriorates late in fermentation after the shift from exponential to constant feed rates, which induces rapid resource depletion and changes in growth rate. In addition, one of the output states of the AND gate failed in the bioreactor, effectively making it only responsive to a single input. Finally, cells carrying the AND gate show considerably less accumulation of biomass. Overall, these results highlight challenges and suggest modified strategies for developing and characterizing genetic circuits that function reliably during fermentation.
Synthetic Biology; Systems Biology; Genetic Compiler; RBS Calculator; Computer-Aided Design; Fermentation
Bacteria construct elaborate nanostructures, obtain nutrients and energy from diverse sources, synthesize complex molecules, and implement signal processing to react to their environment. These complex phenotypes require the coordinated action of multiple genes, which are often encoded in a contiguous region of the genome, referred to as a gene cluster. Gene clusters sometimes contain all of the genes necessary and sufficient for a particular function. As an evolutionary mechanism, gene clusters facilitate the horizontal transfer of the complete function between species. Here, we review recent work on a number of clusters whose functions are relevant to biotechnology. Engineering these clusters has been hindered by their regulatory complexity, the need to balance the expression of many genes, and a lack of tools to design and manipulate DNA at this scale. Advances in synthetic biology will enable the large-scale bottom-up engineering of the clusters to optimize their functions, wake up cryptic clusters, or to transfer them between organisms. Understanding and manipulating gene clusters will move towards an era of genome engineering, where multiple functions can be “mixed-and-matched” to create a designer organism.
Systems Biology; Genetic Parts; Devices; Refactoring; Biotechnology
The interaction specificities of extracytoplasmic function (ECF) sigma (σ) factors with promoters and their negative regulators (anti-σs) were mapped to identify non-crossreacting parts. These orthogonal sets represent a synthetic biology toolbox of genetic switches.
Part mining was applied to characterize 86 extracytoplasmic function (ECF) σs, their promoters, and 62 anti-σs identified from the genomes of diverse bacteria.A subset of 20 σs and promoters were found to be highly orthogonal to each other and can be used to build non-crossreacting switches in single cells.The N- and C-terminal domains from σs from different subgroups can be recombined and recognize the corresponding chimeric promoter.These parts functioned off-the-shelf in an E. coli host with minimal re-engineering and minimally affected host growth and gene expression.
Cells react to their environment through gene regulatory networks. Network integrity requires minimization of undesired crosstalk between their biomolecules. Similar constraints also limit the use of regulators when building synthetic circuits for engineering applications. Here, we mapped the promoter specificities of extracytoplasmic function (ECF) σs as well as the specificity of their interaction with anti-σs. DNA synthesis was used to build 86 ECF σs (two from every subgroup), their promoters, and 62 anti-σs identified from the genomes of diverse bacteria. A subset of 20 σs and promoters were found to be highly orthogonal to each other. This set can be increased by combining the −35 and −10 binding domains from different subgroups to build chimeras that target sequences unrepresented in any subgroup. The orthogonal σs, anti-σs, and promoters were used to build synthetic genetic switches in Escherichia coli. This represents a genome-scale resource of the properties of ECF σs and a resource for synthetic biology, where this set of well-characterized regulatory parts will enable the construction of sophisticated gene expression programs.
compiler; genetic circuit; part mining; synthetic biology; systems biology
Two-component systems enable bacteria to sense changes in their environment and adjust gene expression in response. Multiple two-component systems could function as a combinatorial sensor to discriminate environmental conditions. A combinatorial sensor is composed of a set of sensors that are non-specifically activated to different magnitudes by many stimuli, such that their collective activity pattern defines the signal. Using promoter reporters and flow cytometry, we measured the response of three two-component systems in Escherichia coli that have been previously reported to respond to many environmental stimuli (EnvZ/OmpR, CpxA/CpxR, and RcsC/RcsD/RcsB). A chemical library was screened for the ability to activate the sensors and 13 inducers were identified that produce different patterns of sensor activity. The activities of the three systems are uncorrelated with each other and the osmolarity of the inducing media. Five of the seven possible non-trivial patterns generated by three sensors are observed. This data demonstrate one mechanism by which bacteria are able to use a limited set of sensors to identify a diverse set of compounds and environmental conditions.
systems biology; synthetic biology; membrane fluidity; butanol; isobutanol; biofuel; biodiesel; stress response
Synthetic genetic sensors and circuits enable programmable control over the timing and conditions of gene expression. They are being increasingly incorporated into the control of complex, multigene pathways and cellular functions. Here, we propose a design strategy to genetically separate the sensing/circuitry functions from the pathway to be controlled. This separation is achieved by having the output of the circuit drive the expression of a polymerase, which then activates the pathway from polymerase-specific promoters. The sensors, circuits and polymerase are encoded together on a ‘controller’ plasmid. Variants of T7 RNA polymerase that reduce toxicity were constructed and used as scaffolds for the construction of four orthogonal polymerases identified via part mining that bind to unique promoter sequences. This set is highly orthogonal and induces cognate promoters by 8- to 75-fold more than off-target promoters. These orthogonal polymerases enable four independent channels linking the outputs of circuits to the control of different cellular functions. As a demonstration, we constructed a controller plasmid that integrates two inducible systems, implements an AND logic operation and toggles between metabolic pathways that change Escherichia coli green (deoxychromoviridans) and red (lycopene). The advantages of this organization are that (i) the regulation of the pathway can be changed simply by introducing a different controller plasmid, (ii) transcription is orthogonal to host machinery and (iii) the pathway genes are not transcribed in the absence of a controller and are thus more easily carried without invoking evolutionary pressure.
Many applications require cells to switch between discrete phenotypic states. Here, we harness the FimBE inversion switch to flip a promoter, allowing expression to be toggled between two genes oriented in opposite directions. The response characteristics of the switch are characterized using two-color cytometry. This switch is used to toggle between orthogonal chemosensory pathways by controlling the expression of CheW and CheW*, which interact with the Tar (aspartate) and Tsr* (serine) chemoreceptors, respectively. CheW* and Tsr* each contain a mutation at their protein-protein interface such that they interact with each other. The complete genetic program containing an arabinose-inducible FimE controlling CheW/CheW* (and constitutively-expressed tar/tsr*) is transformed into an E. coli strain lacking all native chemoreceptors. This program enables bacteria to swim towards serine or aspartate in the absence or presence of arabinose, respectively. Thus, the program functions as a multiplexer with arabinose as the selector. This demonstrates the ability of synthetic genetic circuits to connect to a natural signaling network to switch between phenotypes.
genetic memory; recombinase; stochastic switching; synthetic biology; systems biology
Light is a powerful tool for manipulating living cells because it can be applied with high resolution across space and over time. We previously constructed a red-light sensitive E. coli transcription system based on a chimera between the red/far red switchable cyanobacterial phytochrome Cph1 and the E. coli EnvZ/OmpR two-component signaling pathways. Here we report the development of a green light inducible transcription system in E. coli based on a recently discovered green/red photoswitchable two-component system from cyanobacteria. We demonstrate that transcriptional output is proportional to the intensity of green light applied and that the green sensor is orthogonal to the red sensor at intensities of 532nm light less than 0.01W/m2. Expression of both sensors in a single cell allows two-color optical control of transcription in both batch culture and in patterns across a lawn of engineered cells. Because each sensor functions as a photoreversible switch, this system should allow the spatial and temporal control of the expression of multiple genes though different combinations of light wavelengths. This feature should aid precision single cell and population-level studies in systems and synthetic biology.
Light-regulated promoter; synthetic biology; two-component system; phytochrome; cyanobacteriochrome
Optogenetic modules offer cell biologists unprecedented new ways to poke and prod cells. The combination of these precision perturbative tools with observational tools, such as fluorescent proteins, may dramatically accelerate our ability to understand the inner workings of the cell.
The increasing scale and sophistication of genetic engineering will necessitate a new generation of computer-aided design (CAD). For large genetic programs, keeping track of the DNA on the level of nucleotides becomes tedious and error prone. To push the size of projects, it is important to abstract the designer from the process of part selection and optimization. The vision is to specify genetic programs in a higher-level language, which a genetic compiler could automatically convert into a DNA sequence. Steps towards this goal include: defining the semantics of the higher-level language, algorithms to select and assemble parts, and biophysical methods to link DNA sequence to function. These will be coupled to graphic design interfaces and simulation packages to aid in the prediction of program dynamics, optimize genes, and scan projects for errors.
Computer-aided design; systems biology; synthetic biology; design automation
Genetically-encodable optical reporters, such as Green Fluorescent Protein, have revolutionized the observation and measurement of cellular states. However, the inverse challenge of using light to precisely control cellular behavior has only recently begun to be addressed; semi-synthetic chromophore-tethered receptors1 and naturally-occurring channel rhodopsins have been used to directly perturb neuronal networks2,3. The difficulty of engineering light sensitive proteins remains a significant impediment to the optical control to most cell-biological processes. Here we demonstrate the use of a new genetically-encoded light-control system based on an optimized reversible protein-protein interaction from the phytochrome signaling network of Arabidopsis thaliana. Because protein-protein interactions are one of the most general currencies of cellular information, this system can in principal be generically used to control diverse functions. Here we show that this system can be used to precisely and reversibly translocate target proteins to the membrane with micrometer spatial resolution and second time resolution. We show that light-gated translocation of the upstream activators of rho-family GTPases, which control the actin cytoskeleton, can be used to precisely reshape and direct the cell morphology of mammalian cells. The light-gated protein-protein interaction that has been optimized in this work should be useful for the design of diverse light-programmable reagents, potentially enabling a new generation of perturbative, quantitative experiments in cell biology.
Two-component systems are a class of sensors that enable bacteria to respond to environmental and cell-state signals. The canonical system consists of a membrane-bound sensor histidine kinase that autophosphorylates in response to a signal and transfers the phosphate to an intracellular response regulator. Bacteria typically have dozens of two-component systems. The key questions are whether these systems are linear and, if they are, how cross talk between systems is buffered. In this work, we studied the EnvZ/OmpR and CpxA/CpxR systems from Escherichia coli, which have been shown previously to exhibit slow cross talk in vitro. Using in vitro radiolabeling and a rapid quenched-flow apparatus, we experimentally measured 10 biochemical parameters capturing the cognate and non-cognate phosphotransfer reactions between the systems. These data were used to parameterize a mathematical model that was used to predict how cross talk is affected as different genes are knocked out. It was predicted that significant cross talk between EnvZ and CpxR only occurs for the triple mutant ΔompR ΔcpxA ΔactA-pta. All seven combinations of these knockouts were made to test this prediction and only the triple mutant demonstrated significant cross talk, where the cpxP promoter was induced 280-fold upon the activation of EnvZ. Furthermore, the behavior of the other knockouts agrees with the model predictions. These results support a kinetic model of buffering where both the cognate bifunctional phosphatase activity and the competition between regulator proteins for phosphate prevent cross talk in vivo.
two-component systems; systems biology; synthetic biology; computational biology; genetic circuits
The type III secretion system (T3SS) is a molecular machine in gram negative bacteria that exports proteins through both membranes to the extracellular environment. It has been previously demonstrated that the T3SS encoded in Salmonella Pathogenicity Island 1 (SPI-1) can be harnessed to export recombinant proteins. Here, we demonstrate the secretion of a variety of unfolded spider silk proteins and use these data to quantify the constraints of this system with respect to the export of recombinant protein.
To test how the timing and level of protein expression affects secretion, we designed a hybrid promoter that combines an IPTG-inducible system with a natural genetic circuit that controls effector expression in Salmonella (psicA). LacO operators are placed in various locations in the psicA promoter and the optimal induction occurs when a single operator is placed at the +5nt (234-fold) and a lower basal level of expression is achieved when a second operator is placed at -63nt to take advantage of DNA looping. Using this tool, we find that the secretion efficiency (protein secreted divided by total expressed) is constant as a function of total expressed. We also demonstrate that the secretion flux peaks at 8 hours. We then use whole gene DNA synthesis to construct codon optimized spider silk genes for full-length (3129 amino acids) Latrodectus hesperus dragline silk, Bombyx mori cocoon silk, and Nephila clavipes flagelliform silk and PCR is used to create eight truncations of these genes. These proteins are all unfolded polypeptides and they encompass a variety of length, charge, and amino acid compositions. We find those proteins fewer than 550 amino acids reliably secrete and the probability declines significantly after ~700 amino acids. There also is a charge optimum at -2.4, and secretion efficiency declines for very positively or negatively charged proteins. There is no significant correlation with hydrophobicity.
We show that the natural system encoded in SPI-1 only produces high titers of secreted protein for 4-8 hours when the natural psicA promoter is used to drive expression. Secretion efficiency can be high, but declines for charged or large sequences. A quantitative characterization of these constraints will facilitate the effective use and engineering of this system.
Edge detection is a signal processing algorithm common in artificial intelligence and image recognition programs. We have constructed a genetically encoded edge detection algorithm that programs an isogenic community of E.coli to sense an image of light, communicate to identify the light-dark edges, and visually present the result of the computation. The algorithm is implemented using multiple genetic circuits. An engineered light sensor enables cells to distinguish between light and dark regions. In the dark, cells produce a diffusible chemical signal that diffuses into light regions. Genetic logic gates are used so that only cells that sense light and the diffusible signal produce a positive output. A mathematical model constructed from first principles and parameterized with experimental measurements of the component circuits predicts the performance of the complete program. Quantitatively accurate models will facilitate the engineering of more complex biological behaviors and inform bottom-up studies of natural genetic regulatory networks.
Microbial engineering often requires fine control over protein expression; for example, to connect genetic circuits 1-7 or control flux through a metabolic pathway 8-13. We have developed a predictive design method for synthetic ribosome binding sites that enables the rational control of a protein's production rate on a proportional scale. Experimental validation of over 100 predictions in Escherichia coli shows that the method is accurate to within a factor of 2.3 over a range of 100,000-fold. The design method also correctly predicts that reusing a ribosome binding site sequence in different genetic contexts can result in different protein expression levels. We demonstrate the method's utility by rationally optimizing a protein's expression level to connect a genetic sensor to a synthetic circuit. The proposed forward engineering approach will accelerate the construction and systematic optimization of large genetic systems.
synthetic biology; translation; optimization; metabolic engineering; genetic circuit; RNA secondary structure
The type III secretion system (T3SS) exports proteins from the cytoplasm, through both the inner and outer membranes, to the external environment. Here, a system is constructed to harness the T3SS encoded within Salmonella Pathogeneity Island 1 to export proteins of biotechnological interest. The system is composed of an operon containing the target protein fused to an N-terminal secretion tag and its cognate chaperone. Transcription is controlled by a genetic circuit that only turns on when the cell is actively secreting protein. The system is refined using a small human protein (DH domain) and demonstrated by exporting three silk monomers (ADF-1, -2, and -3), representative of different types of spider silk. Synthetic genes encoding silk monomers were designed to enhance genetic stability and codon usage, constructed by automated DNA synthesis, and cloned into the secretion control system. Secretion rates up to 1.8 mg l−1 h−1 are demonstrated with up to 14% of expressed protein secreted. This work introduces new parts to control protein secretion in Gram-negative bacteria, which will be broadly applicable to problems in biotechnology.
automated DNA synthesis; cellular engineering; biomaterials; genetic parts; systems biology