Establishing a High-Yield Chloroplast Cell-Free System for Prototyping Genetic Parts.

Plastid engineering offers the potential to carry multigene traits in plants; however, it requires reliable genetic parts to balance expression. The difficulty of chloroplast transformation and slow plant growth makes it challenging to build plants just to characterize genetic parts. To address these limitations, we developed a high-yield cell-free system from Nicotiana tabacum chloroplast extracts for prototyping genetic parts. Our cell-free system uses combined transcription and translation driven by T7 RNA polymerase and works with plasmid or linear template DNA. To develop our system, we optimized lysis, extract preparation procedures (e.g., runoff reaction, centrifugation, and dialysis), and the physiochemical reaction conditions. Our cell-free system can synthesize 34 ± 1 µg/mL luciferase in batch reactions and 60 ± 4 µg/mL in semicontinuous reactions. We apply our batch reaction system to test a library of 103 ribosome binding site (RBS) variants and rank them based on cell-free gene expression. We observe a 1300-fold dynamic range of luciferase expression when normalized by maximum mRNA expression, as assessed by the malachite green aptamer. We also find that the observed normalized gene expression in chloroplast extracts and the predictions made by the RBS Calculator are correlated. We anticipate that chloroplast cell-free systems will increase the speed and reliability of building genetic systems in plant chloroplasts for diverse applications.

Analysis of Poly(ethylene terephthalate) degradation kinetics of evolved IsPETase variants using a surface crowding model.

Poly(ethylene terephthalate) (PET) is a major plastic polymer utilized in the single-use and textile industries. The discovery of PET-degrading enzymes (PETases) has led to an increased interest in the biological recycling of PET in addition to mechanical recycling. IsPETase from Ideonella sakaiensis is a candidate catalyst, but little is understood about its structure-function relationships with regards to PET degradation. To understand the effects of mutations on IsPETase productivity, we develop a directed evolution assay to identify mutations beneficial to PET film degradation at 30 °C. IsPETase also displays enzyme concentration-dependent inhibition effects, and surface crowding has been proposed as a causal phenomenon. Based on total internal reflectance fluorescence microscopy and adsorption experiments, IsPETase is likely experiencing crowded conditions on PET films. Molecular dynamics simulations of IsPETase variants reveal a decrease in active site flexibility in free enzymes and reduced probability of productive active site formation in substrate-bound enzymes under crowding. Hence, we develop a surface crowding model to analyze the biochemical effects of three hit mutations (T116P, S238N, S290P) that enhanced ambient temperature activity and/or thermostability. We find that T116P decreases susceptibility to crowding, resulting in higher PET degradation product accumulation despite no change in intrinsic catalytic rate. In conclusion, we show that a macromolecular crowding-based biochemical model can be used to analyze the effects of mutations on properties of PETases and that crowding behavior is a major property to be targeted for enzyme engineering for improved PET degradation.

Systematic mining of the human microbiome identifies antimicrobial peptides with diverse activity spectra.

Human-associated bacteria secrete modified peptides to control host physiology and remodel the microbiota species composition. Here we scanned 2,229 Human Microbiome Project genomes of species colonizing skin, gastrointestinal tract, urogenital tract, mouth and trachea for gene clusters encoding RiPPs (ribosomally synthesized and post-translationally modified peptides). We found 218 lanthipeptides and 25 lasso peptides, 70 of which were synthesized and expressed in E. coli and 23 could be purified and functionally characterized. They were tested for activity against bacteria associated with healthy human flora and pathogens. New antibiotics were identified against strains implicated in skin, nasal and vaginal dysbiosis as well as from oral strains selectively targeting those in the gut. Extended- and narrow-spectrum antibiotics were found against methicillin-resistant Staphylococcus aureus and vancomycin-resistant Enterococci. Mining natural products produced by human-associated microbes will enable the elucidation of ecological relationships and may be a rich resource for antimicrobial discovery.

Redesign of an Escherichia coli Nissle treatment for phenylketonuria using insulated genomic landing pads and genetic circuits to reduce burden.

To build therapeutic strains, Escherichia coli Nissle (EcN) have been engineered to express antibiotics, toxin-degrading enzymes, immunoregulators, and anti-cancer chemotherapies. For efficacy, the recombinant genes need to be highly expressed, but this imposes a burden on the cell, and plasmids are difficult to maintain in the body. To address these problems, we have developed landing pads in the EcN genome and genetic circuits to control therapeutic gene expression. These tools were applied to EcN SYNB1618, undergoing clinical trials as a phenylketonuria treatment. The pathway for converting phenylalanine to trans-cinnamic acid was moved to a landing pad under the control of a circuit that keeps the pathway off during storage. The resulting strain (EcN SYN8784) achieved higher activity than EcN SYNB1618, reaching levels near when the pathway is carried on a plasmid. This work demonstrates a simple system for engineering EcN that aids quantitative strain design for therapeutics.

Functional expression of diverse post-translational peptide-modifying enzymes in Escherichia coli under uniform expression and purification conditions.

RiPPs (ribosomally-synthesized and post-translationally modified peptides) are a class of pharmaceutically-relevant natural products expressed as precursor peptides before being enzymatically processed into their final functional forms. Bioinformatic methods have illuminated hundreds of thousands of RiPP enzymes in sequence databases and the number of characterized chemical modifications is growing rapidly; however, it remains difficult to functionally express them in a heterologous host. One challenge is peptide stability, which we addressed by designing a RiPP stabilization tag (RST) based on a small ubiquitin-like modifier (SUMO) domain that can be fused to the N- or C-terminus of the precursor peptide and proteolytically removed after modification. This is demonstrated to stabilize expression of eight RiPPs representative of diverse phyla. Further, using Escherichia coli for heterologous expression, we identify a common set of media and growth conditions where 24 modifying enzymes, representative of diverse chemistries, are functional. The high success rate and broad applicability of this system facilitates: (i) RiPP discovery through high-throughput "mining" and (ii) artificial combination of enzymes from different pathways to create a desired peptide.

Selection for constrained peptides that bind to a single target protein.

Peptide secondary metabolites are common in nature and have diverse pharmacologically-relevant functions, from antibiotics to cross-kingdom signaling. Here, we present a method to design large libraries of modified peptides in Escherichia coli and screen them in vivo to identify those that bind to a single target-of-interest. Constrained peptide scaffolds were produced using modified enzymes gleaned from microbial RiPP (ribosomally synthesized and post-translationally modified peptide) pathways and diversified to build large libraries. The binding of a RiPP to a protein target leads to the intein-catalyzed release of an RNA polymerase σ factor, which drives the expression of selectable markers. As a proof-of-concept, a selection was performed for binding to the SARS-CoV-2 Spike receptor binding domain. A 1625 Da constrained peptide (AMK-1057) was found that binds with similar affinity (990 ± 5 nM) as an ACE2-derived peptide. This demonstrates a generalizable method to identify constrained peptides that adhere to a single protein target, as a step towards "molecular glues" for therapeutics and diagnostics.

Coculture of primary human colon monolayer with human gut bacteria.

The presence of microbes in the colon impacts host physiology. Therefore, microbes are being evaluated as potential treatments for colorectal diseases. Humanized model systems that enable robust culture of primary human intestinal cells with bacteria facilitate evaluation of potential treatments. Here, we describe a protocol that can be used to coculture a primary human colon monolayer with aerotolerant bacteria. Primary human colon cells maintained as organoids are dispersed into single-cell suspensions and then seeded on collagen-coated Transwell inserts, where they attach and proliferate to form confluent monolayers within days of seeding. The confluent monolayers are differentiated for an additional 4 d and then cocultured with bacteria. As an example application, we describe how to coculture differentiated colon cells for 8 h with four strains of Bacteroides thetaiotaomicron, each engineered to detect different colonic microenvironments via genetically embedded logic circuits incorporating deoxycholic acid and anhydrotetracycline sensors. Characterization of this coculture system reveals that barrier function remains intact in the presence of engineered B. thetaiotaomicron. The bacteria stay close to the mucus layer and respond in a microenvironment-specific manner to the inducers (deoxycholic acid and anhydrotetracycline) of the genetic circuits. This protocol thus provides a useful mucosal barrier system to assess the effects of bacterial cells that respond to the colonic microenvironment, and may also be useful in other contexts to model human intestinal barrier properties and microbiota-host interactions.

Synthetic biology open language visual (SBOL Visual) version 2.3.

People who are engineering biological organisms often find it useful to communicate in diagrams, both about the structure of the nucleic acid sequences that they are engineering and about the functional relationships between sequence features and other molecular species. Some typical practices and conventions have begun to emerge for such diagrams. The Synthetic Biology Open Language Visual (SBOL Visual) has been developed as a standard for organizing and systematizing such conventions in order to produce a coherent language for expressing the structure and function of genetic designs. This document details version 2.3 of SBOL Visual, which builds on the prior SBOL Visual 2.2 in several ways. First, the specification now includes higher-level "interactions with interactions," such as an inducer molecule stimulating a repression interaction. Second, binding with a nucleic acid backbone can be shown by overlapping glyphs, as with other molecular complexes. Finally, a new "unspecified interaction" glyph is added for visualizing interactions whose nature is unknown, the "insulator" glyph is deprecated in favor of a new "inert DNA spacer" glyph, and the polypeptide region glyph is recommended for showing 2A sequences.

An absorbance method for analysis of enzymatic degradation kinetics of poly(ethylene terephthalate) films.

Increased interest in poly(ethylene terephthalate) (PET)-degrading enzymes (PETases) have generated efforts to find mutants with improved catalytic activity and thermostability. Here, we present a simple and fast method to determine relative enzyme kinetics through bulk absorbance measurements of released products over time. A thermostable variant of PETase from Ideonella sakaiensis was engineered (R280A S121E D186H N233C S282C) with a denaturation temperature of 69.4 ± 0.3 °C. This was used to assess the method's ability to determine relative enzyme kinetics across variants and reveal structure-function relationships. Measurements at 24 and 72 h at 400 nM of enzyme suggest that the mutations improved catalytic rates 5- to 7-fold. On the contrary, kinetic analyses of the thermostable variant and wild-type reveal different reaction trajectories despite similar maximum catalytic rates, resulting in higher product accumulation from the thermostable variant over time. The results of the assay support the necessity for kinetic measurements to determine relationships between sequence and function for IsPETase and other PET hydrolases.

Nanoliter scale electrochemistry of natural and engineered electroactive bacteria.

Bacterial extracellular electron transfer (EET) is envisioned for use in applied biotechnologies, necessitating electrochemical characterization of natural and engineered electroactive biofilms under conditions similar to the target application, including small-scale biosensing or biosynthesis platforms, which is often distinct from standard 100 mL-scale stirred-batch bioelectrochemical test platforms used in the laboratory. Here, we adapted an eight chamber, nanoliter volume (500 nL) electrochemical flow cell to grow biofilms of both natural (Biocathode MCL community, Marinobacter atlanticus, and Shewanella oneidensis MR1) or genetically modified (S. oneidensis ΔMtr and S. oneidensis ΔMtr + pLB2) electroactive bacteria on electrodes held at a constant potential. Maximum current density achieved by unmodified strains was similar between the nano- and milliliter-scale reactors. However, S. oneidensis biofilms engineered to activate EET upon exposure to 2,4-diacetylphloroglucinol (DAPG) produced current at wild-type levels in the stirred-batch reactor, but not in the nanoliter flow cell. We hypothesize this was due to differences in mass transport of DAPG, naturally-produced soluble redox mediators, and oxygen between the two reactor types. Results presented here demonstrate, for the first time, nanoliter scale chronoamperometry and cyclic voltammetry of a range of electroactive bacteria in a three-electrode reactor system towards development of miniaturized, and potentially high throughput, bioelectrochemical platforms.

Synthetic biology open language visual (SBOL visual) version 2.2.

People who are engineering biological organisms often find it useful to communicate in diagrams, both about the structure of the nucleic acid sequences that they are engineering and about the functional relationships between sequence features and other molecular species. Some typical practices and conventions have begun to emerge for such diagrams. The Synthetic Biology Open Language Visual (SBOL Visual) has been developed as a standard for organizing and systematizing such conventions in order to produce a coherent language for expressing the structure and function of genetic designs. This document details version 2.2 of SBOL Visual, which builds on the prior SBOL Visual 2.1 in several ways. First, the grounding of molecular species glyphs is changed from BioPAX to SBO, aligning with the use of SBO terms for interaction glyphs. Second, new glyphs are added for proteins, introns, and polypeptide regions (e. g., protein domains), the prior recommended macromolecule glyph is deprecated in favor of its alternative, and small polygons are introduced as alternative glyphs for simple chemicals.

Genetic Encoding of Targeted Magnetic Resonance Imaging Contrast Agents for Tumor Imaging.

Tumor-selective contrast agents have the potential to aid in the diagnosis and treatment of cancer using noninvasive imaging modalities such as magnetic resonance imaging (MRI). Such contrast agents can consist of magnetic nanoparticles incorporating functionalities that respond to cues specific to tumor environments. Genetically engineering magnetotactic bacteria to display peptides has been investigated as a means to produce contrast agents that combine the robust image contrast effects of magnetosomes with the transgenic-targeting peptides displayed on their surface. This work reports the first use of magnetic nanoparticles that display genetically encoded pH low insertion peptide (pHLIP), a long peptide intended to enhance MRI contrast by targeting the extracellular acidity associated with the tumors. To demonstrate the modularity of this versatile platform to incorporate diverse targeting ligands by genetic engineering, we also incorporated the cyclic αv integrin-binding peptide iRGD into separate magnetosomes. Specifically, we investigate their potential for enhanced binding and tumor imaging both in vitro and in vivo. Our experiments indicate that these tailored magnetosomes retain their magnetic properties, making them well suited as T2 contrast agents, while exhibiting an increased binding compared to the binding in wild-type magnetosomes.

Bacterial terpene biosynthesis: challenges and opportunities for pathway engineering.

Terpenoids are the largest and structurally most diverse class of natural products. They possess potent and specific biological activity in multiple assays and against diseases, including cancer and malaria as notable examples. Although the number of characterized terpenoid molecules is huge, our knowledge of how they are biosynthesized is limited, particularly when compared to the well-studied thiotemplate assembly lines. Bacteria have only recently been recognized as having the genetic potential to biosynthesize a large number of complex terpenoids, but our current ability to associate genetic potential with molecular structure is severely restricted. The canonical terpene biosynthetic pathway uses a single enzyme to form a cyclized hydrocarbon backbone followed by modifications with a suite of tailoring enzymes that can generate dozens of different products from a single backbone. This functional promiscuity of terpene biosynthetic pathways renders terpene biosynthesis susceptible to rational pathway engineering using the latest developments in the field of synthetic biology. These engineered pathways will not only facilitate the rational creation of both known and novel terpenoids, their development will deepen our understanding of a significant branch of biosynthesis. The biosynthetic insights gained will likely empower a greater degree of engineering proficiency for non-natural terpene biosynthetic pathways and pave the way towards the biotechnological production of high value terpenoids.

Biosynthesis of the nitrogenase active-site cofactor precursor NifB-co in Saccharomyces cerevisiae.

The radical S-adenosylmethionine (SAM) enzyme NifB occupies a central and essential position in nitrogenase biogenesis. NifB catalyzes the formation of an [8Fe-9S-C] cluster, called NifB-co, which constitutes the core of the active-site cofactors for all 3 nitrogenase types. Here, we produce functional NifB in aerobically cultured Saccharomyces cerevisiae Combinatorial pathway design was employed to construct 62 strains in which transcription units driving different expression levels of mitochondria-targeted nif genes (nifUSXB and fdxN) were integrated into the chromosome. Two combinatorial libraries totaling 0.7 Mb were constructed: An expression library of 6 partial clusters, including nifUSX and fdxN, and a library consisting of 28 different nifB genes mined from the Structure-Function Linkage Database and expressed at different levels according to a factorial design. We show that coexpression in yeast of the nitrogenase maturation proteins NifU, NifS, and FdxN from Azotobacter vinelandii with NifB from the archaea Methanocaldococcus infernus or Methanothermobacter thermautotrophicus yields NifB proteins equipped with [Fe-S] clusters that, as purified, support in vitro formation of NifB-co. Proof of in vivo NifB-co formation was additionally obtained. NifX as purified from aerobically cultured S. cerevisiae coexpressing M. thermautotrophicus NifB with A. vinelandii NifU, NifS, and FdxN, and engineered yeast SAM synthase supported FeMo-co synthesis, indicative of NifX carrying in vivo-formed NifB-co. This study defines the minimal genetic determinants for the formation of the key precursor in the nitrogenase cofactor biosynthetic pathway in a eukaryotic organism.

Deep learning to predict the lab-of-origin of engineered DNA.

Genetic engineering projects are rapidly growing in scale and complexity, driven by new tools to design and construct DNA. There is increasing concern that widened access to these technologies could lead to attempts to construct cells for malicious intent, illegal drug production, or to steal intellectual property. Determining the origin of a DNA sequence is difficult and time-consuming. Here deep learning is applied to predict the lab-of-origin of a DNA sequence. A convolutional neural network was trained on the Addgene plasmid dataset that contained 42,364 engineered DNA sequences from 2230 labs as of February 2016. The network correctly identifies the source lab 48% of the time and 70% it appears in the top 10 predicted labs. Often, there is not a single "smoking gun" that affiliates a DNA sequence with a lab. Rather, it is a combination of design choices that are individually common but collectively reveal the designer.

A Pressure Test to Make 10 Molecules in 90 Days: External Evaluation of Methods to Engineer Biology.

Centralized facilities for genetic engineering, or "biofoundries", offer the potential to design organisms to address emerging needs in medicine, agriculture, industry, and defense. The field has seen rapid advances in technology, but it is difficult to gauge current capabilities or identify gaps across projects. To this end, our foundry was assessed via a timed "pressure test", in which 3 months were given to build organisms to produce 10 molecules unknown to us in advance. By applying a diversity of new approaches, we produced the desired molecule or a closely related one for six out of 10 targets during the performance period and made advances toward production of the others as well. Specifically, we increased the titers of 1-hexadecanol, pyrrolnitrin, and pacidamycin D, found novel routes to the enediyne warhead underlying powerful antimicrobials, established a cell-free system for monoterpene production, produced an intermediate toward vincristine biosynthesis, and encoded 7802 individually retrievable pathways to 540 bisindoles in a DNA pool. Pathways to tetrahydrofuran and barbamide were designed and constructed, but toxicity or analytical tools inhibited further progress. In sum, we constructed 1.2 Mb DNA, built 215 strains spanning five species ( Saccharomyces cerevisiae, Escherichia coli, Streptomyces albidoflavus, Streptomyces coelicolor, and Streptomyces albovinaceus), established two cell-free systems, and performed 690 assays developed in-house for the molecules.

Post-translational control of genetic circuits using Potyvirus proteases.

Genetic engineering projects often require control over when a protein is degraded. To this end, we use a fusion between a degron and an inactivating peptide that can be added to the N-terminus of a protein. When the corresponding protease is expressed, it cleaves the peptide and the protein is degraded. Three protease:cleavage site pairs from Potyvirus are shown to be orthogonal and active in exposing degrons, releasing inhibitory domains and cleaving polyproteins. This toolbox is applied to the design of genetic circuits as a means to control regulator activity and degradation. First, we demonstrate that a gate can be constructed by constitutively expressing an inactivated repressor and having an input promoter drive the expression of the protease. It is also shown that the proteolytic release of an inhibitory domain can improve the dynamic range of a transcriptional gate (200-fold repression). Next, we design polyproteins containing multiple repressors and show that their cleavage can be used to control multiple outputs. Finally, we demonstrate that the dynamic range of an output can be improved (8-fold to 190-fold) with the addition of a protease-cleaved degron. Thus, controllable proteolysis offers a powerful tool for modulating and expanding the function of synthetic gene circuits.

Genetic encoding of DNA nanostructures and their self-assembly in living bacteria.

The field of DNA nanotechnology has harnessed the programmability of DNA base pairing to direct single-stranded DNAs (ssDNAs) to assemble into desired 3D structures. Here, we show the ability to express ssDNAs in Escherichia coli (32-205 nt), which can form structures in vivo or be purified for in vitro assembly. Each ssDNA is encoded by a gene that is transcribed into non-coding RNA containing a 3'-hairpin (HTBS). HTBS recruits HIV reverse transcriptase, which nucleates DNA synthesis and is aided in elongation by murine leukemia reverse transcriptase. Purified ssDNA that is produced in vivo is used to assemble large 1D wires (300 nm) and 2D sheets (5.8 µm(2)) in vitro. Intracellular assembly is demonstrated using a four-ssDNA crossover nanostructure that recruits split YFP when properly assembled. Genetically encoding DNA nanostructures provides a route for their production as well as applications in living cells.

Advances in genetic circuit design: novel biochemistries, deep part mining, and precision gene expression.

Cells use regulatory networks to perform computational operations to respond to their environment. Reliably manipulating such networks would be valuable for many applications in biotechnology; for example, in having genes turn on only under a defined set of conditions or implementing dynamic or temporal control of expression. Still, building such synthetic regulatory circuits remains one of the most difficult challenges in genetic engineering and as a result they have not found widespread application. Here, we review recent advances that address the key challenges in the forward design of genetic circuits. First, we look at new design concepts, including the construction of layered digital and analog circuits, and new approaches to control circuit response functions. Second, we review recent work to apply part mining and computational design to expand the number of regulators that can be used together within one cell. Finally, we describe new approaches to obtain precise gene expression and to reduce context dependence that will accelerate circuit design by more reliably balancing regulators while reducing toxicity.