Engineered dCas9 with reduced toxicity in bacteria: implications for genetic circuit design

Large synthetic genetic circuits require the simultaneous expression of many regulators. Deactivated Cas9 (dCas9) can serve as a repressor by having a small guide RNA (sgRNA) direct it to bind a promoter. The programmability and specificity of RNA:DNA basepairing simplifies the generation of many orthogonal sgRNAs that, in theory, could serve as a large set of regulators in a circuit. However, dCas9 is toxic in many bacteria, thus limiting how high it can be expressed, and low concentrations are quickly sequestered by multiple sgRNAs. Here, we construct a non-toxic version of dCas9 by eliminating PAM (protospacer adjacent motif) binding with a R1335K mutation (dCas9*) and recovering DNA binding by fusing it to the PhlF repressor (dCas9*_PhlF). Both the 30 bp PhlF operator and 20 bp sgRNA binding site are required to repress a promoter. The larger region required for recognition mitigates toxicity in Escherichia coli, allowing up to 9600 ± 800 molecules of dCas9*_PhlF per cell before growth or morphology are impacted, as compared to 530 ± 40 molecules of dCas9. Further, PhlF multimerization leads to an increase in average cooperativity from n = 0.9 (dCas9) to 1.6 (dCas9*_PhlF). A set of 30 orthogonal sgRNA-promoter pairs are characterized as NOT gates; however, the simultaneous use of multiple sgRNAs leads to a monotonic decline in repression and after 15 are co-expressed the dynamic range is <10-fold. This work introduces a non-toxic variant of dCas9, critical for its use in applications in metabolic engineering and synthetic biology, and exposes a limitation in the number of regulators that can be used in one cell when they rely on a shared resource.United States. Defense Advanced Research Projects Agency (DARPA HR0011-15-C-0084 Living Foundries: 1000 Molecules Program

Targeted DNA degradation using a CRISPR device stably carried in the host genome

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 10[superscript 8]-fold. When the genome is targeted, this causes cell death, reducing viable cells by a factor of 10[superscript 8]. 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.National Science Foundation (U.S.). Synthetic Biology Engineering Research Center (SA5284-11210)United States. Defense Advanced Research Projects Agency (Contract N66001-12-C-4187

Dynamic behavior of the monomer–monomer surface reaction model with adsorbate interactions

The monomer–monomer surface reaction model with an adsorbate interaction term is studied. An epidemic analysis of the poisoning times (tp)(tp) for small square lattices as a function of lattice edge length LL and interaction strength α at the point of equal adsorption rates yields a dynamic scaling relation which describes the crossover between log-power-law and exponential behavior in L,L, and is able to fit the entire dependence of tptp upon α and L.L. The phase transition is further explored by varying adsorption rates and is found to follow second-order kinetics. A mean-field approximation is introduced as a comparison for the numerical results. © 1997 American Institute of Physics.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/70692/2/JCPSA6-107-18-7397-1.pd

Quantification of the physiochemical constraints on the export of spider silk proteins by Salmonella type III secretion

<p>Abstract</p> <p>Background</p> <p>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 <it>Salmonella </it>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.</p> <p>Results</p> <p>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 <it>Salmonella </it>(<it>psicA</it>). LacO operators are placed in various locations in the <it>psicA </it>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) <it>Latrodectus hesperus </it>dragline silk, <it>Bombyx mori </it>cocoon silk, and <it>Nephila clavipes </it>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.</p> <p>Conclusions</p> <p>We show that the natural system encoded in SPI-1 only produces high titers of secreted protein for 4-8 hours when the natural <it>psicA </it>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.</p

Multi-input CRISPR/Cas genetic circuits that interface host regulatory networks

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 σ[subscript 70] 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).United States. Defense Advanced Research Projects Agency (CLIO N66001‐12‐C‐4016)National Institutes of Health (U.S.) (GM095765)National Institute of General Medical Sciences (U.S.) (Grant P50 GMO98792)Synthetic Biology Engineering Research Center (EEC0540879)United States. Defense Advanced Research Projects Agency (Ginkgo BioWorks. CLIO N66001‐12‐C‐4018)United States. Office of Naval Research. Multidisciplinary University Research Initiative (Grant N00014‐13‐1‐0074)United States. Office of Naval Research. Multidisciplinary University Research Initiative (Boston University. Award 4500000552)United States. Air Force Office of Scientific Research (FA9550‐11‐C‐0028)American Society for Engineering Education. National Defense Science and Engineering Graduate Fellowship (32 CFR 168a

Assessing the effect of dynamics on the closed-loop protein-folding hypothesis

The closed-loop (loop-n-lock) hypothesis of protein folding suggests that loops of about 25 residues, closed through interactions between the loop ends (locks), play an important role in protein structure. Coarse-grain elastic network simulations, and examination of loop lengths in a diverse set of proteins, each supports a bias towards loops of close to 25 residues in length between residues of high stability. Previous studies have established a correlation between total contact distance (TCD), a metric of sequence distances between contacting residues (cf. contact order), and the log-folding rate of a protein. In a set of 43 proteins, we identify an improved correlation ( r 2 = 0.76), when the metric is restricted to residues contacting the locks, compared to the equivalent result when all residues are considered ( r 2 = 0.65). This provides qualified support for the hypothesis, albeit with an increased emphasis upon the importance of a much larger set of residues surrounding the locks. Evidence of a similar-sized protein core/extended nucleus (with significant overlap) was obtained from TCD calculations in which residues were successively eliminated according to their hydrophobicity and connectivity, and from molecular dynamics simulations. Our results suggest that while folding is determined by a subset of residues that can be predicted by application of the closed-loop hypothesis, the original hypothesis is too simplistic; efficient protein folding is dependent on a considerably larger subset of residues than those involved in lock formation. </jats:p

Engineering PET-degrading enzymes for biorecycling and bioremediation

Plastics, due to their inert properties and resistance to biodegradation, have been ravaging ecosystems worldwide and are especially harmful to aquatic wildlife. Plastics in the environment wear and tear into micron sized particles, termed microplastics, which are ingested and/or affect organisms at every level of the food chain. Recently, microplastics have been reported in human feces, and their potential health hazards remain unknown. In 2010, 4.8-12.7 million metric tons entered the oceans due to mismanagement and leakage, with an additional 31 million metric tons when terrestrial and freshwater ecosystems are considered. Both food and water supplies are likely contaminated with microplastics, and we need technologies to decrease formation of microplastics and remove these particulates from the environment. One of the most synthesized plastics is poly(ethylene terephthalate) (PET), an aromatic polyester with extremely low degradation rates. Due to the huge negative environmental impact of PET products, efficient recycling strategies need to be designed to “close the loop”’ to reduce dependence on petroleum feedstocks and decrease economic loss through single-use practices. The recent discovery of a PET-consuming bacteria Ideonella sakaiensis and its PET hydrolases has shown potential for enzyme-mediated recycling and bioremediation. Here, we present preliminary characterization of the catalytic rate of the newly discovered PETase and its behaviour over time, with perspective into future engineering potential for the enzyme for use in industrial processes

Genetic Sensor for Strong Methylating Compounds

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 S[subscript N]1 and S[subscript N]2 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.National Science Foundation (U.S.). Graduate Research FellowshipUnited States. Defense Advanced Research Projects Agency. Chronical of Lineage Indicative of Origins (N66001-12-C-4018)United States. Office of Naval Research (N00014-10-1-0245)United States. Office of Naval Research (N00014-13-1-0074)National Science Foundation (U.S.) (557686-2117)National Science Foundation (U.S.). Synthetic Biology Engineering Research Center (SA5284-11210

Environmental signal integration by a modular AND gate

Microorganisms use genetic circuits to integrate environmental information. We have constructed a synthetic AND gate in the bacterium Escherichia coli that integrates information from two promoters as inputs and activates a promoter output only when both input promoters are transcriptionally active. The integration occurs via an interaction between an mRNA and tRNA. The first promoter controls the transcription of a T7 RNA polymerase gene with two internal amber stop codons blocking translation. The second promoter controls the amber suppressor tRNA supD. When both components are transcribed, T7 RNA polymerase is synthesized and this in turn activates a T7 promoter. Because inputs and outputs are promoters, the design is modular; that is, it can be reconnected to integrate different input signals and the output can be used to drive different cellular responses. We demonstrate this modularity by wiring the gate to integrate natural promoters (responding to Mg2+ and AI-1) and using it to implement a phenotypic output (invasion of mammalian cells). A mathematical model of the transfer function is derived and parameterized using experimental data