Symposium SM03—Growing Next-Generation Materials with Synthetic Biology

I will describe approaches to obtain new materials from engineered cells and how genetic engineering can be applied to change the material properties. The focus will be on materials that are difficult or impossible to produce via synthetic chemistry and have unique functional properties (e.g., electrical or optical).

Bacterial cellulose is an exopolysaccharide produced by various species of bacteria, with Gluconacetobacter xylinus being the model organism to decipher the biosynthesis of cellulose. It has potential applications in food, pharmaceutical, and cosmetic industries due to its unique structure and mechanical property. The cellulose produced by bacteria is of high purity and crystallinity compared to those isolated from wood pulp due to the absence of lignin and hemicellulose. Despite the natural production capacity and the biogenesis knowledge, bacterial cellulose is produced in relatively low quantity. In this study, we systematically rewire the natural production cascade to enhance cellulose production capacity and the self-assembly of microfibrils for the synthesis of tailor-made and customizable cellulose. Gluconacetobacter xylinus produces cellulose by converting UDP-glucose to cellulose through cellulose synthase located in the cell membrane. The gene encoding cellulose synthase is comprised of four subunits and is arranged in an operon. To study the metabolism of genetically modified cellulose synthase and how it affect cellulose production, we develop genetic tools and build a library of genetic parts in BioBrick formats. We have completed the construction of genetic toolkit library for the manipulation of three Acetobacteraceae family members that are Gluconacetobacter xylinus (ATCC 700178), Gluconacetobacter hansenii (ATCC 53582), Komagataeibacter rhaeticus (iGEM). We further constructed plasmids carrying different combinations of the four cellulose synthase subunits and introduced them into the bacteria for expression to create a mutant library of synthetic host capable of producing cellulose with different capacity and property.
In this study, we also examine the self-assembly mechanisms and microstructures of bacterial cellulose. Never-dried native bacterial cellulose produced from two Acetobacteraceae family members are disintegrated into individual fibres through TEMPO-mediated oxidation and mechanical treatment, obtaining transparent and viscous suspension. Although the main oxidant is NaClO, we find that mechanical treatment (ultrasonication) plays an important role in the disintegration process of the bacterial cellulose. The morphology of the disintegrated fibrils are observed by SEM and the crystallinity of the disintegrated fibers are analysed by XRD. The organic functional group of the cellulose fibres as are characterized in dry state and wet condition by FTIR and RAMAN, respectively. The birefringence of the fibre suspension is also investigated under polarizing microscope. The oxidized cellulose fibres are expected to have physical interaction with metal ions. We will study this interaction in respect of forming conductive cellulose complexation. We further develop a method to synthesize bacterial cellulose hollow microparticles that are challenging to achieve using conventional method. The reformatted cellulose have been shown to improve wound healing“

We have developed a system for fabricating and perturbing self-healing oligonucleotide-based attractor patterns in photolithographically-patterned hydrogels using ultraviolet radiation. Digital maskless photolithography combined with microfluidic handling methods provides the ability to construct soft materials of arbitrary shape at biologically relevant length scales of tens to hundreds of microns in a matter of seconds. The photoinitiator system we incorporate with this platform enables the UV-light directed initiation of photosensitive DNA-based reactions in structured hydrogels. We use this approach to first form self-healing linear and hill-shaped DNA-based patterns in poly(ethylene-glycol) diacrylate hydrogels. Undisturbed, these spatial patterns were stable over at least 24 hours. When patterns were perturbed in particular areas they reformed their original shape. The ability to grow and perturb gradients of biomolecules such as DNA may facilitate the study of cell populations in dynamic microenvironments, providing experimentalists control over where and when chemical cues are induced or inhibited at the microscale.

Harnessing and engineering natural biological processes to synthesize and self-assemble highly ordered materials with nanoscale components and features is key for developing the next generation of functional biomaterials. One such nanoscale material naturally synthesized by some bacteria is a self-assembling, selectively-permeable protein shell called a bacterial microcompartment (BMC). These microcompartments are used by cells to shield sensitive metabolic reactions from the intracellular environment (e.g. oxygen sensitive CO2 fixation enzymes or enzymes that produce toxic intermediates). The selectively permeable shell consists of different protein components, most with a pore at the center, which serves to concentrate reactants and increase reaction kinetics. BMCs are an attractive platform because the components of the microcompartments (the shell proteins and/or the encapsulated proteins) can be engineered, synthesized separately, and self-assembled in the absence of a living cell, providing a great deal of design space for customization. As a proof of principle, a one class of microcompartment shell protein (the trimeric BMC-T protein) was engineered to coordinate a FeS metal cluster at the pore, which conferred reversible redox activity to the protein in solution. Further, the FeS-modified BMC-T protein also displayed reversible redox activity when it was bound to an electrode surface, with a similar formal potential (Eo’ = –370 mV vs SHE) to what was obtained in solution. Further, when the protein was engineered to coordinate the FeS cluster to the opposite side of the pore, multiple redox peaks with different midpoint potentials (Eo’ = –460 and –340 mV vs SHE) were observed. We have also observed redox activity from an electrode-bound BMC-T protein modified to coordinate a Cu metal center at the pore, with a formal potential(s) that is also dependent on the site modified to coordinate the metal center (Eo’ = 200 mV [site 1] vs. 410 and 550 mV [site 2] vs SHE). These results suggest that the BMC platform is a versatile architecture that can be customized to suit various applications (e.g. sensor development, electrosynthesis, and energy storage and conversion) and represent an important step toward harnessing naturally-occurring, self-assembling, nanoscale biomaterials to perform surface bound redox chemistry in a tunable, controlled environment.

Bio-based chemicals currently receive attention as sustainable and drop-in substitutes for petroleum-based chemicals. The feedstocks used for the production of bio-based chemicals have recently expanded from edible sugars to inedible lignocellulosic biomass ⁽¹⁾. In the last decade, recent progress in metabolic engineering has expanded fermentation products from aliphatic compounds to aromatic compounds, which can be used in a vast array of industrial applications, including the production of chemicals, medications, and electronics ⁽²⁾. In addition, synthetic biology enables the production of non-natural aromatics, including phenylpropanoids and chorismate derivatives. This diversity is crucial to expand the development and industrial uses of bio-based chemicals to produce bio-based polymers, food additives, and pharmaceuticals, in addition to building block chemicals for biorefinery ⁽³⁾. Most of bio-based chemicals are currently produced from edible sugars or starches that compete directly with food and feed uses. Technologies that will enable utilization of inedible and renewable lignocellulosic feedstocks are needed to realize a bio-based economy. To achieve the goal, we aimed at developing a microbial platform and tailoring the platform to desired compounds by creating artificial pathways in Escherichia coli cells with metabolic engineering and synthetic biology. Resulting microbial cell factories produced the derivatives of chorismate and aromatic amino acids from glucose or renewable lignocellulosic feedstock, such as kraft pulp and sorghum bagasse ⁽⁴⁾. In addition, future perspectives and challenges in this research field will be presented.

Keywords: Biorefinery; Lignocellulosic biomass; Metabolic engineering; Synthetic biology.

References:
Kawaguchi, H. Hasunuma, T. Ogino, C. Kondo, A. (2016) Bioprocessing of bio-based chemicals produced from lignocellulosic feedstocks. Curr Opin Biotechnol. 42:30–39.
Kawaguchi, H. Ogino, C. Kondo, A. (2017) Microbial conversion of biomass into bio-based polymers. Bioresour Technol. 245(Part B):1664-1673.
Noda, S. Kondo, A. (2017) Recent advances in microbial production of aromatic chemicals and derivatives. Trends Biotechnol. 35(8):785-796.
Noda, S., Shirai, T., Oyama, S., Kondo, A. (2016) Metabolic design of a platform Escherichia coli strain producing various chorismate derivatives. Metab Eng. 33:119-129.

Biology is unparalleled in its molecular diversity and wide application space, especially in novel materials. There are however numerous challenges associated with realizing the full potential of this molecular diversity. Optimization of microbial production for any biological target requires repeated iterations of the design-build-test-analyze engineering cycle. The rate at which any team can execute the engineering cycle directly affects the development time for any product. Similarly, the magnitude of strain improvement in each cycle impacts overall costs and development time. At Amyris, advanced tools for strain engineering, high throughput screening, analytics, and bioinformatics have been developed over the years to accelerate the engineering cycle and reduce the number of necessary iterations. The presentation will cover details of the automated platforms that enable Amyris scientists to rapidly cycle through a data-driven strain improvement process. It will also illustrate the important role that Amyris’ biotechnology is playing today with industry, academia, and the government, in providing a sustainable, cost-effective alternative to traditional manufacturing practices for producing new materials.

Development of silicon-based materials with highly organized nano- and microstructures are of increasing demand across the medical, optical, energy, and mechanical fields. Nanoscale control of silica fabrication is desirable as new functions and properties can emerge from complex structures. Current methods for controlling silica morphology often require chemical additives, extremes of temperature, pressure, or pH, and long processing times. Interestingly, biological organisms, like diatoms, naturally produce a diversity of hierarchical silica structures under ambient conditions that are difficult to obtain even through advanced synthetic methods. Here, we use the silica-mineralizing R5 peptide from the diatom Cylindrotheca fusiformis in combination with targeted post-translational modifications (PTMs) to rapidly synthesize silica with controlled morphology at room temperature in aqueous conditions. By screening a diverse library of modifying enzymes for activity against R5, we identified ten enzymes capable of introducing PTMs to R5 and describe a set of design rules that relate individual modifications to specific changes in silica morphology. We then use these rules to rationally design and express synthetic enzyme pathways containing multiple modifying enzymes to selectively modify R5, and demonstrate that multiple modifications create synergistic effects on silica morphology. Additionally, we show that R5 can be used in combination with synthetic and biologically produced metal nanoparticles to synthesize silica-coated core-shell nanoparticles with tunable shell thickness. This system provides a modular approach for rapidly engineering and synthesizing silica structures under ambient conditions and moves us closer to being able to synthesize complex silica structures, like those seen in nature, that will provide new functions and properties to help advance silicon-based application spaces.

In recent years, metallic nanoparticles are being used for biomedical, environmental, and industrial applications. Metallic nanoparticles, such as gold nanoparticles (AuNPs) feature prominently in plasmonics, biosensing, photoacoustic and photothermal imaging. Polydopamine, which can lead to the formation of eumelanin is a biomolecule which has also gained lot of interest due to its broad absorption spectrum and has potential for optical, photo-acoustic, and biosensing applications. Formation of a biocomposite material combining such optically-active materials could lead to a tunable absorption spectrum, based on polydopamine thickness.

As the complexity of composite materials increases, the development of simple design rules become essential, and using biological building blocks is one method to achieve this. Bacterial synthesis routes offer immense potential for an efficient synthesis process, ease of functionalization, modification and repeatability. Genetically modified E Coli strains can synthesize extracellular curli nanofibers, which is an amyloid fibrous structural components present in biofilms. We have designed and assembled DNA constructs to produce curli fibers with the ability to reduce gold nanoparticles and a polydopamine-binding fusion protein. Two E. coli strains were transformed with the constructs to produce the melanin coated well-organized gold nanoparticles. Curli fibers were assembled with alternating structural curli protein, CsgA (repeat unit) segments and one of the CsgA units was conjugated to spy tag protein, while the other was conjugated to the peptide fragment (FlgA3) which reduces gold nanoparticles from the auric chloride solution. A fusion protein comprised of the spycatcher protein conjugated to the repeat unit of the Pmel17 amyloid fiber was created and the fusion lead to the formation of the gold nanoparticle-polydopamine conjugate. The goal of this study is to design and produce novel polydopamine-bound gold nanoparticles using synthetic biology approaches, to generate a preliminary model for future investigation of the feasibility and advantages of bacterially-produced tunable biocomposite materials, which could find applications in plasmonics and catalysis.

Synthetic biology has garnered interest as a tool for the production of synthetically challenging materials. Recently, a facile bio-catalytic route to the synthesis of molecules containing carbon-silicon bonds has been reported, potentially connecting synthetic biology with the production of silicon-based materials. In this work, we report on our recent efforts towards the creation of a synthetic biology system to generate a wide range of novel, silicon containing polymers to be used as precursors for the production of robust, high-temperature materials. Ultimately, our goals are to leverage synthetic biology to create silicon containing precursor materials in an environmentally friendly, scalable manner, and to take advantage of the remarkable ability of biomolecules to self-assemble at multiple length scales to enhance the functional capabilities of the resulting high-temperature materials.

The emerging field of synthetic biology provides opportunity and new capabilities for the precision synthesis, assembly, and function of architected materials. Currently, there are significant challenges for the integration of bio-derived and bio-based technologies in the defense-oriented materials space, in which materials are expected to perform under broad operating conditions. Army platforms must reliably operate over wide temperature ranges (e.g. -40 to 75 C) and extremes in humidity. In addition, materials must exhibit the durability to be fielded for long periods of time (e.g. 5+ years) and endure strain rate deformations from low strain, high frequency vibration to high strain impact events. We anticipate a significant challenge in harnessing synthetic biology to develop materials that are effective across this environmental breadth, particularly when considering that automated/parallelized biological experiments are not aligned to traditional materials science characterization strategies that typically occur in series and require much larger scale of material. This submission will give an overview of current projects employing the tools of Synthetic Biology and highlight needs of materials in context of the Materials & Manufacturing Science Division of the Army Research Laboratory.

Nature has acquired a vast repertoire of catalytic functionality. As the diversity of this repertoire is perpetually expanded upon by evolution’s process of random mutation and selection, a similar method may be employed to repurpose existing enzymes for novel function. Enzymes and their engineered variants have relevance where current production routes are lacking; they have the ability to improve product yield, materials price, supply chain security and/or environmental waste accumulation. This work aims to develop platforms for generating enzyme variants in order to facilitate production of molecules with desirable economic and industrial properties. As a starting point, we are investigating the screening and selection of halogenating enzymes for use in materials production. Several classes of halogenase are being examined for the ability to install chlorine, bromine, and fluorine into carbon frameworks. Our presented work highlights a single class of enzyme, the vanadium-dependent haloperoxidase, which displays a broad substrate reactivity profile, stability in organic solvent, and ease of recombinant expression and purification. In addition to performing bromination and chlorination, these haloperoxidases have also been shown to perform other potentially useful reactions such as hydroxylation, sulfoxidation, and epoxidation. We hope to eventually explore the manipulation of this enzyme and its reaction conditions to direct it towards the catalysis of specific substrates and chemical transformations.

Phloroglucinol (PG) is a precursor to TATB which is an energetic chemical of interest to the DoD. PG is part of the ‘Pressure Test’ as selected by DoD stakeholders for the MIT-Broad Foundry and is a compelling test case for the Tri-Service Synthetic Biology for Military Environment (SBME) research efforts. The Tri-Service Laboratories approached the PG single ‘challenge problem’ by leveraging SBME capabilities such as strain engineering capabilities, strain banks, and rapid prototyping of paper-based sensors. The AFRL team has an aircraft and fuel isolate library enriched for Pseudomonads, which are known to harbor PG synthesizing enzymes known as PhlD. The AFRL team has designed selective PhlD primers for screening Pseudomonad isolates for diverse and potentially more active PhlD enzymes than found in current literature. The NRL team is taking advantage of Marinobacter CP1’s (SBME chassis) ability to accumulate significant amounts of the PG precursor malonyl-CoA. Marinobacter will be genetically engineered to produce PG through a combination of genome editing to divert carbon to malonyl-CoA and the subsequent conversion to PG through the addition of a plasmid- expressed PhlD. The ARL team is harnessing the robust SBME chassis Clostridium acetobutylicum to model and optimize its capability to produce significant amounts of the PG precursor malonyl-CoA with plasmid-expressed PhlD. ARL has determined that mixed sugar inputs yield optimal PG production. Additionally, ARL will construct a living PG sensor in soil microorganisms using ICE technology obtained from the Voigt laboratory. The ECBC team and Northwestern University collaborators have developed and optimized a paper-based, cell-free PG sensor. The paper-based PG detection system will be used in field-deployable conditions where the sensor would be triggered by PG, resulting in rapid reporting of the presence of PG. The MIT-Broad Foundry has optimized PG production in the model chassis E. coli and has built a PG genetic sensor for rapid screening. The PG Tri-Service collaborative efforts in biological and cell-free systems will result in optimized PG production and detection, invaluable for DoD and synthetic biology applications.

Conventionally, in vitro protein synthesis has been focused on a top-down approach when effective transcription/translation process relied on compartmentalization of the whole cell-free system in liposomal vesicles. In this method, having an appropriate phospholipid membrane fluidity played important role not only in the progression of protein expression, but also in mechanical stability of a very rigid osmotic system. Alternatively, protein-based vesicles prepared using bottom-up approach can solve some of the hurdles associated with liposomal vesicles. Assembled in a layered fashion, microcapsules afford tailored membrane porosity, variable DNA loading capacity and improved mechanical and chemical stability necessary for a prolonged shelf storage of RNA-based aptamers. We demonstrated that during layer-by-layer assembly, several DNA templates encoding various RNA-based aptamers and riboswitches can be immobilized in microcapsules prepared from reconstituted silk fibroin protein. Per activation of RNA aptamers with a specific analyte, DNA templates immobilized in microcapsules were proven to be functional on transcriptional and translational levels. During incubation in a cell-free system, the rate of expression of a reporter gene was similar to the rate of expression for microcapsule-bound DNA templates, confirming that the functionality of aptamers was conserved. By increasing the loading capacity of DNA templates in microcapsules, the level of gene expression can be amplified. Further functionalization of silk shells with gold nanoparticles and antibodies will allow to expand multi-component DNA- microcapsule complex design for targeted surveillance of several biomarkers. The proposed design of multifunctional protein-based microcapsules provide necessary robustness to RNA-based aptamers for their long-term storage or prolonged circulation, as microcapsule-bound DNA templates have been proven to be functional after one year storage at ambient conditions.

This research is funded by the Air Force Office of Scientific Research (AFOSR).

Plasmonic metal nanoparticles have interesting physiochemical properties that have been used for many applications including biosensors, cellular imaging and drug delivery. Conjugation between gold nanoparticles (AuNP) and biomolecules, proteins and nucleic acids expand these applications. Here, we applied the Polymerase Chain Reaction (PCR) to build a genetic circuit based on an oligo-conjugated-AuNP foundation. Constructed gene networks are created using amplicons containing a T7 promoter followed by an open reading frame for a gene of interest and a T7 terminator. Our current focus is on optimizing PCR reaction for creating gold-conjugated gene networks as well as characterization of the AuNP-DNA complexes by Transmission Electron Microscopy (TEM) and Atomic Force Microscopy (AFM). With this approach, we plan to expand potential applications for the constructed gene networks by coupling the networks to cell free protein expression systems and exploring plasmonic tunability of gene expression.

Microbial gas vesicles (GVs) are gas-filled protein microstructures encoded by biosynthetic pathways endogenous to marine bacteria and extremophiles and function to provide a way to control buoyancy by the host organism. Composed of a single layer of protein, gas vesicles are remarkable in that they exclude water from their interiors despite forming in an aqueous environment. To explore materials applications of gas vesicles, we have transported a 10 gene GV operon onto an inducible expression vector and recapitulated vesicle assembly in E. coli cells. E.coli cells producing gas vesicles are further incorporated into hydrogels to create materials with tunable density that can be used to make environmentally-responsive biomaterials with variable acoustic properties. Further, we envision and are working towards altering the genetic control of the system to make GV-expression responsive to additional inducer signals and subsequently bio-print GV-expressing cells into gel-matrices to enable spatio-temporal manipulation of hydrogel properties.

While the field of biology seeks to understand the functions and mechanisms of natural organisms, synthetic biology aims to use biological functions to engineer materials and systems. This engineering view is at the core of synthetic biology and seeks to define fundamental building blocks in nature that can be manipulated in a variety of combinations to design and produce complex systems. To illustrate this approach Ball compared² the components and the level of complexity that goes into the design and manufacturing of an automobile to the process of biological cell formation – building materials (glass, metal vs nucleotides, amino acids); components (pistons, wheels vs proteins); modules (microcontroller vs genetic circuits), etc.

Barnatt¹ asked a thought provoking question related to synthetic biology: “will synthetic acorns grow into biobuildings?” There are many aspects that need to be considered to begin to answer the question: 1. what materials that are needed for this quest; 2. what is the level of complexity involved; 3. what are associated building costs, are they competitive. While this rhetorical question draws attention to the possibilities of synthetic biology, a rapidly growing community is exploring nearer-term applications (synthetic biochemical production, biocomputing, biosensors, etc).

To partially answer this question we have initiated a study to characterize the complexity required to create engineered organic systems. While complexity is a relative term, computer software development can be a suitable benchmark due to similarities between instruction storing function of DNA and binary code. Further similarities between organic and software systems are the use of limited serial instruction sets (quaternary or binary) to produce arbitrarily complex outcomes when executed on deterministic systems.

The starting point is the comparison of the genome of different organisms to landmark software programming projects. The number of base pairs in the genome of organisms³ ranges from ~ 1 to 10 million for bacteria to ~ 1 billion for birds to a few billions for humans and other mammals. Major software projects for which some details (number of lines of code) are available⁴ have been selected for comparison, including the Hubble Space Telescope, the Mars Curiosity Rover, the Android smart phone operating system. The extent of compiled object code ranges from several hundred million to a few billion characters. The similarity between numbers of software characters in complex computer programs and base pairs in genomes, suggests that programming complex synthetic biosystems, while quite challenging and requiring a major effort, is likely to be feasible.

Biological coding provides additional functionality beyond storing of genetic information. Molecular structure and shape provide the means for reproduction of the genetic code, its transformation into amino acids and proteins, etc. This represents the storage of additional (2^nd, 3^rd order) valuable information that needs to be accounted in future analysis. It is important to note that genome size that does not necessarily correlate with organism complexity. Interestingly, a similar case may be made for software programs, in that their size is not only related to the complexity of the system function but also on the computer language used, the period when the programming was carried out, other needs of the system, etc. Nonetheless, our preliminary evaluation indicates that the level of complexity involved in the genome of even the most sophisticated organism is comparable to large-scale man-made computer programs.

1. C. Barnatt, Synthetic Biology, Jan. 2018.
2. P. Ball, Synthetic Biology-Engineering nature to make materials, MRS Bulletin, 43 (7), 477-484, July 2018.
3. R. Milo and R. Phillips, Cell Biology by the Numbers, 2015, Garland Science, Taylor & Francis.
4. T. Wendt, et al., Consolidation in Vehicle Electronic Architectures, 2015, Roland Berger Strategy Consultants.

Cell-free protein synthesis (CFPS) is a promising approach for the production of biomaterials that cannot be synthesized by existing organic chemistry techniques. CFPS avoids many of the pitfalls of bacterial expression of biomaterials by allowing direct access to the chemical reaction that is taking place. Among the advantages of CFPS are increased tolerance to toxic reagents and products, facile incorporation of non-biological components such as abiotic amino-acids, the absence of cell transfection or protein purification steps, and increased yield as the CFPS system only makes the proteins it has been templated to produce. NMR monitoring of CFPS would allow us to fine-tune these systems so that they may be successfully scaled in a cost-effective manner. CFPS systems are commonly optimized via the production of green-fluorescent protein (GFP) which is spectroscopically monitored as the reaction proceeds. This approach fails in anaerobic environments since GFP chromophore maturation is oxygen dependent. Further, GFP fusions are not applicable for the production of small molecules. By utilizing radioisotope labeled substrates and various 1D and 2D techniques we expect to be able to monitor the utilization of substrates, generation of the desired products, and flux of related metabolites of various CFPS reactions. Significant differences in CFPS yield have been observed when reactions are prepared by different users and/or different cell lysates. NMR spectroscopy may be able to elucidate the mechanism behind this reaction variability. We have been able to identify metabolic differences between lysates prepared by different users. When the reactants and cell lysate used in CFPS are controlled, NMR metabolite profiles differ between users. The production of side products, such as ethanol and lactic acid, varies by user and by lysate utilized. In-situ NMR may be able to identify mechanisms for low product yield, discover undesirable metabolism pathways that are being activated, or if energy deficits are occurring.

Utilization of various photosensitizers in metal-organic frameworks (MOFs) for photocatalytic oxidation of toxic compounds has been of recent interest. Typically these MOFs incorporate pyrene, porphyrins, or organometallic photosensitizers into the organic linker of the MOF. However, through the utilization of solvent assisted ligand incorporation (SALI), a wide variety of compounds can be post-synthetically incorporated into the 3 nm pores of NU-1000. We report here the incorporation of protoporphyrin IX produced in E. coli via the Shemin pathway into NU-1000. Various porphyrins can be produced via the Shemin pathway including copropyrphyrin and uroporphyrin. Each of these porphyrins have carboxylic acid handles that can be incorporated into NU-1000 via SALI. The inclusion of these porphyrins into NU-1000 improved the rate of photocatalytic degradation of the MOF toward the chemical warfare agent simlulant for sulfur mustard, 2-chloroethyl ethyl sulfide. Utilization of synthetic biology in MOF synthesis opens up new pathways that are yet to be realized through the production of novel or difficult to synthesize ligands.

Imagine a world where the buildings in which we live, work, and play — the walls, the roofs, the floors, the windows — are grown, maintained, and healed autonomously by living microbial communities. Imagine architects of the future that swap their sketchbooks for genetic tools that encode the architecture of a building right into the DNA of those communities. Living architecture may soon be more than a vision penned in a science fiction novel. In fact, we are closer to realizing that vision than you think, thanks to interdisciplinary teams of researchers armed with a commitment — and the synthetic biology toolkits — to make that vision a reality.
Recent advances in metabolic engineering have enabled autonomous, high-fidelity biomanufacturing of useful chemical, mineral, and polymer building blocks that can be leveraged in the design and fabrication of construction materials and living architectures at the human scale. This presentation will highlight the research efforts of a highly interdisciplinary and collaborative team at the University of Colorado Boulder that is integrating synthetic biology, microbiology, materials science, and structural engineering to design and fabricate useful minerals (e.g., aggregates for concrete production) and polymers (e.g., styrene) for materials with commercial applications to building design and construction. In one study, we demonstrate for the first time that calcium carbonate (CaCO₃) mineral aggregates for mortar and concrete can be tailored by modulating the precipitation kinetics through genetic engineering of ureolytic microorganisms. We also demonstrate that useful quantities of styrene for construction material manufacture can be biologically produced via microbial engineering. In this presentation, we will also show that engineered microorganisms can be leveraged in the design of hybrid living building materials that display both biological (i.e., living, regenerative self-healing) and structural (i.e., load-bearing) function. Finally, this talk will highlight the challenges that emerge working across length scales and disciplines, as well as the grand opportunity that exists for synthetic biologists and materials engineers to work together to create never-before-imagined material solutions for critical societal problems in energy, water, and the built environment.

Biological systems have adapted diverse ways to control gene expression. Riboswitches are regulatory elements located in the 5'-untranslated region of mRNA that change the folded structure of the mRNA upon the binding of effector molecule and, as a consequence, alter the expression of the downstream gene. Besides the naturally evolved riboswitches that are triggered by cellular metabolites, several synthetic riboswitches have been developed that can sense and respond to non-endogenous small molecules such as theophylline, tetracycline or 2,4,6-trinitrotoluene (TNT). Majority of the synthetic riboswitches were selected and tested in the cell-based systems, usually E. coli. Cell-free protein expression systems (CFES) have several advantages for the development and testing of synthetic riboswitches including the elimination of the interaction with complex cellular networks and decoupling of transcription and translation processes. We studied the performance of theophylline-based synthetic riboswitch coupled with the sfGFP reporter gene in E.coli cellular extract and PURE cell-free systems. To monitor the mRNA dynamics a malachite green aptamer sequence was added to the 3’-untranslated region of sfGFP. Performance of the theophylline riboswitch was compared with a constitutively expressed sfGFP (control). Transcription dynamics of mRNA with riboswitch was very similar to the transcription of the control mRNA for all theophylline concentrations tested in both E. coli extract and PURE CFES. However, sfGFP expression in riboswitch construct was order of magnitude lower even at highest concentration of theophylline. This indicate that riboswitch altered the accessibility of the ribosome binding site and translation initiation rate. Dose-response curves showed similar behavior in both CFES with high micromollar to low millimolar dynamic range. The experimental effort was accompanied by computational modeling and simulations. A kinetic model of riboswitch activation was developed.

This research is funded by the Air Force Office of Scientific Research (AFOSR).

As the desire for the latest product innovations continues, the need for new materials innovation has becoming a reality. New materials development would be made easier by having access to a new palette of chemical building-blocks, and such a palette of molecules with diverse structure and functionality is already available in nature today. Through the intersection of bioengineering, synthetic chemistry and material science, we at Zymergen are working to bring such novel building blocks to bear on materials innovation. In this presentation we will review our efforts in targeted rapid prototyping of biotarget production, and the development of diverse new materials useful in a variety of advanced product sectors.

Melanin is a pigment ubiquitous in nature and is found in all types of living organisms. Due to its unique physicochemical properties, high biocompatibility and biostability, there has been increasing interest in the use of melanin for bioelectronic, biomedical and theranostic applications. In the vast majority of prior studies, melanin has been either chemically synthesized or isolated from animals, restricting its use to small-scale applications. In this study, we engineered the fast-growing marine bacterium Vibrio natriegens to synthesize melanin by expressing a heterologous tyrosinase gene. The V. natriegens melanin demonstrated characteristic electron paramagnetic resonance signals which were enhanced upon illumination. When melanized cells were immobilized at an electrode surface and probed with diffusible mediators and electrical inputs, we observed that melanization conferred redox activities to V. natriegens. The photoprotective properties of the bacterially-produced melanin were validated by its protection of bacterial and HeLa cells from UV irradiation. Moreover, melanized bacteria were able to quickly adsorb organic compounds such as chloroquine and trinitrotoluene. Providing the engineered V. natriegens strain with tyrosine or tyrosine-containing peptides as precursors led to the production of melanin nanoparticles and variants with varying optical properties, suggesting that this system is tunable and could be exploited for novel materials applications. Overall, the genetic tractability, rapid division time and ease of culture using comparatively inexpensive carbon sources provides a set of attractive properties that compare favorably to current Escherichia coli production strains and warrant the further development of this chassis as microbial factory for natural products synthesis.
Furthermore, we report results demonstrating the potential of Vibrio natriegens produced melanin in chemical protective scenarios. The fabric preparation utilized microwave initiation in the presence of (3-aminopropyl)triethoxysilane to provide primary amine groups on the surface. Analysis of water vapor transport through the melanin-modified fabric indicated that the treatment should have little impact on wearer comfort. The permeation of three chemical agent simulant vapors, 2-chloroethyl ethyl sulfide (CEES), methyl salicylate, and dimethyl methylphosphonate, through the fabric samples was evaluated. Overall, the melanized fabrics demonstrated a reduction in the peak rate and total amount transported for each of the simulants tested with the most dramatic outcome being an 86% reduction in the peak rate and 90% reduction in the total amount of CEES vapor transported through melanin-treated fabric when compared to the untreated fabric. These results suggest that the adsorptive properties of these inexpensive natural polymers can be further exploited and tuned using standard synthetic biology tools to produce additional application relevant compounds. Ongoing work is considering differing melanin materials and their impact when considered in this application.

Melanin, a highly-coordinated polymeric nanoparticle found in natural pigments, has many intriguing properties including protecting organisms from ultra-violet radiation, ability to scavenge reactive species and metal ions, and reported electrical conductivity. Melanin particles isolated from naturally pigmented sources are biologically templated with pre-defined shapes that dictate the optical and protective properties of the melanin assemblies. The tyrosinase enzyme is known to be involved in the biosynthesis of melanin, which lends credence to the use of a synthetic biology approach to engineer tyrosinase for melanin production. In this work, Bacillus megaterium tyrosinase was engineered and expressed recombinantly in Escherichia coli. Two forms of tyrosinase fusions were designed to be secreted through the curli export system and anchored on the cell surfaces. Using these systems, small melanin nanoparticles were able to be produced. In addition, melanin ghosts templated by E. coli cells were synthesized and characterized. Thus the tyrosinase expression, secretion and display capability that will be presented here represents a platform capable of allowing for the production colloidal melanin nanoparticles as well as melanin functionalized surfaces. Future work will investigate tuning the optical and electrical properties of the melanin assemblies through control of size and shape of the superstructure.

The ultimate goal of this work is to design a novel cell signaling circuits that will trigger biochemical pathways in response to the oxygenation state of their environment. For this purpose, we used PAS domains which are sensing elements found in all living organism that respond to environmental stimuli including, light, oxygen, redox state and various metabolites. To create new oxygen sensing genetic circuits, chimeras of the heme iron binding PAS domain from the prokaryotic B. japonicum histidine kinase (FixL) are being engineered with the ability to modulate the enzymatic activity of the mammalian PAS domain containing PAS Kinase (PASK), a protein kinase involved in the control of carbon metabolism and cellular energetics. Molecular simulations, coupled with ultrafast spectroscopic techniques, are being used to support the design and testing of the chimeras. Purified chimeras are also tested for their ability to inhibit PASK kinase activity at various oxygenation states, using a newly developed, non-radiological protein kinase assay. The function of these metazoan derived signaling elements will be tested in a prokaryotic system in order to generate orthogonal signaling pathways for cellular metabolic control.

The Escherichia coli ribosome is a 2.4 MDa molecular machine made up of 57 RNA and protein parts. To support bacterial log phase growth with a 40 minute doubling time, ribosomes must be produced at a rate of ~8 ribosomes per second. Once assembled, these ribosomes can synthesize sequence-defined polymers of amino acids with elongation rates of up to 20 amino acids per second and an accuracy of ~99.99%. The rapid rate of synthesis and incredible catalytic capability motivate efforts to understand the systems biology of ribosome biogenesis and function, as well as to create engineered variants with altered function that are capable of synthesizing new functional materials. As a result, the construction of engineered ribosomes has emerged as a defining challenge in synthetic biology at the materials interface. Unfortunately, the requirement of cell viability severely limits the mutations that can be made to the ribosome. In this presentation, we will discuss our efforts to address this critical limitation both in vivo and in vitro. In vivo, we report the first fully orthogonal ribosome-mRNA system by creating ribosomes with tethered subunits (termed Ribo-T). In vitro, we established a cell-free ribosome construction platform, termed iSAT (integrated synthesis, assembly, and translation of synthetic ribosomes) to build and evolve modified ribosomes in test tubes. As a step towards potential future applications, we also describe efforts to generate synthetic, conjugate nanomaterials with altered self-assembly properties by using site-specifically introduced non canonical amino acids into proteins. Our results set the stage to transform synthetic biology by repurposing the translational apparatus to synthesize novel sequence-controlled polymers and functional biomaterials with an expanded chemistry of life.

Bolt Threads is a start-up focused on delivering biomaterials for fashion and apparel with emphasis on the performance properties that Nature has evolved over millions of years, combined with Nature’s biodegradability and material circularity. Beginning with spider silk polymers, we have built a technology platform for the scalable production of nearly any protein-based fiber. Based on their protein sequence, these fibers can be designed for custom mechanical and functional properties. We have recently expanded our platform to include our bioleather— Mylo – made from mycelium (mushroom roots.) Under controlled growth and other process conditions, we grow Mylo to replicate a variety of leathers on the market. We are scaling up the production of Microsilk, Mylo, and more materials into commercial quantities. Bolt Threads continues to look to Nature for inspiration and will be launching more new materials in the coming years.

Biomaterials based on living cells embedded into hydrogel matrices enable a number of advanced applications including biosensing, wound healing, and bioremediation. When the embedding medium consists of hydrogels comprised substantially of proteins, a wide range of functionalizations are possible including in situ functionalizations in response to local stimuli picked up by embedded cells. These responses can be used to modulate material characteristics, produce biologically active dimensions to the hydrogel matrix, or to report on the presence of analytes. To realize these advanced biomaterials, it is essential that embedded cells maintain viability and that the hydrogel matrices offer favorable processing characteristics. In this work, we present efforts to embed microbial cells into protein-based hydrogels. Our goals are to maintain cell responsivity to inducer stimuli and to cast the hydrogels into useful embodiments. We present the results of embedding genetically engineered E. coli into hydrogels comprised of silk and Nvjp1. Nvjp1, a protein originating in marine sandworm jaws, is especially interesting in that its mechanical properties can be modulated over several orders of magnitude through reversible coordination with specific metal ions and thus hydrogels incorporating Nvjp1 could form a strong foundation for mechanically adaptive biomaterials. In this work, we characterize the mechanical properties of cell-embedded hydrogels using microrheology based on differential dynamic microscopy (DDM). DDM is a particle tracking microrheological technique that does not require trajectory segmentation and thus is potentially more amenable to high throughput implementations. Cell survivability and responsivity data are determined using various in situ induction and viability assays and are considered in conjunction with the mechanical properties of the resultant hydrogels in order to determine optimum cell encapsulation formulations and procedures.

Genetic circuits in living cells offer tremendous opportunities for tackling a number of societal problems, from energy, to environment, to medicine. Yet, our ability to design sophisticated systems that function as intended is challenged by context-dependence, wherein the behavior of a circuit depends on the context. Context includes direct connectivity to other modules and the bare presence of other circuits in the same cell. In fact, in vivo circuits all compete with each other for a limited supply of cellular resources, such as RNA polymerases and ribosomes. This competition for resources creates unintended couplings among circuit components, which often disrupt a circuit’s function. In this talk, I will describe a design-oriented modeling framework to capture these effects along with general engineering solutions based on feedback control to mitigate them. I will, in particular describe our recent work on implementing integral controllers through sRNA silencing, which allow any genetic circuit to adapt to changes in translational resources, the main bottleneck for bacterial circuits. Our solution enables scalable design of complex circuits that are robust to fluctuations in ribosome concentration and hence substantially reduces context-dependence of bacterial genetic circuits.

The Army is putting living materials work in an agile way, harnessing nature’s ability to assemble complex materials across scale towards high value assets such as low cost manufacturing of energetics and optical materials via synthetic biology. This includes low energy, sustainable solutions driving the ability to repair and produce at the point-of-need, use of indigenous or sustainable feed stocks, and leap-ahead advances in biohybrid materials and technology providing smart systems providing new protection and sensing capability in operationally challenging and contested environments. With increasing complexity and control the long-term vision is to develop advanced materials imparting living function for operation in Army relevant environments-disruptive capabilities, such as self-healing, adaptation, protection & situational awareness.
The continual convergence of synthetic biology with other fields and disciplines such as additive manufacturing, lab-on-a chip, machine learning, and multi-scale computing promises to usher in a new age of manufacturing, leading to structural and multifunctional materials. Further, as our ability to control and sustain biological systems and understanding and control of self-assembly improves, we can begin to take these advances out of the laboratory to on-demand applications. To accomplish this, key components of our approach includes the development of advanced discovery to manufacturing pipelines, bringing both an understanding of structure-function relationships and ultimately rational design principles along with tools to unlock broad ranges of hosts for access to new material sets and sustainment in military environments. Bringing together materials and application experts across the physical and engineering sciences as well as government, academia and industry will significantly advance our ability to build discovery to product pipelines, with an emphasis up front on concept of operation, manufacturability, and logistics/sustainment.
This paper will present a discussion of the challenges facing this burgeoning field and snapshot of the Army’s synthetic biology program including more detailed discussion of genetic control of the biological/materials science interface.

A route to advanced multifunctional materials is to embed them with living cells that can perform sensing, chemical production, energy scavenging, and actuation. A challenge in realizing this potential is that the conditions for keeping cells alive are not conducive to materials processing and require a continuous source of water and nutrients. Here, we present a 3D printer that can mix material and cell streams in a novel printhead and build 3D objects (up to 2.5 cm by x 1 cm by 1 cm). Hydrogels are printed using 5% agarose, which has a melting temperature (65^oC) consistent with thermophilic cells, a rigid storage modulus (G’= 6.5 x 10^4), exhibits shear thinning, and can be rapidly hardened upon cooling to preserve structural features. Spores of B. subtilis PY79 are printed within the material and germinate on its exterior, including spontaneously in cracks and new surfaces exposed by tears. By introducing genetically engineered bacteria, the materials can sense chemicals (IPTG, aTc, xylose, or vanillic acid) and produce biopolymers (melanin). Further, we show that the spores are resilient to extreme environmental stresses, including desiccation, solvents (ethanol), high osmolarity (1.5 mM NaCl), 365 nm UV light, X-rays, and g-radiation (2.6 kGy). The introduction of a metabolic pathway for melanin improves resistance to 254 nm UV light and g-radiation. The construction of 3D printed materials containing spores enables the living functions to be used for applications that require long-term storage, in-field functionality, or exposure to uncertain environmental stresses.

Hemozoin, a crystalline form of heme generated by malaria-causing parasites, is an attractive alternative to other inorganic oxidizers for energetic materials due to a higher output when combined with nano aluminum (Slocik, J. et al. Small. 2015, 3539.). Developing methods to control hemozoin crystal morphology should enhance current energetic capabilities. Histidine-rich proteins have been shown to promote hemozoin crystallization with sizes smaller than those obtained from synthetic methods. Here, we identify Nvjp1, a jaw protein from sea worm, as a capable catalyst of hemozoin formation. We also compare crystals nucleated from Nvjp1 to those of known hemozoin proteins from Plasmodium. Ultimately, the ability to generate modular nanoenergetic materials will serve present and future military needs.