Thrust 1: Stimuli-Responsive Biosynthetic Materials
Incorporation of genetically engineered cells into polymer composites can overcome limitations of conventional stimuli-responsive polymers. Genes can be activated (or deactivated) by many different stimuli—light, chemicals, heat, mechanical force, the circadian cycle, or a stimulus of the researcher’s choosing. Once activated, these microbes can produce a variety of chemical products, such as enzymes, small molecules, polymers and therapeutic proteins. Incorporating a suite of different microbial components into a single material can lead to complex materials that respond to several inputs with multivariate outputs. Examples of materials that we propose to develop are those that: 1) respond to chemical threats or pollutants by upregulating decontamination enzymes, 2) produce materials that cycle as thermal insulators by synthesizing and degrading biofilms with insulating properties in response to day/night cycles, and 3) express therapeutic proteins (e.g., insulin in response to glucose) and small molecules (e.g., antibiotics in response to bacterial colonization) in response to disease states.
Thrust 1 will combine polymer synthesis/fabrication with genetic engineering to derive materials that are biosynthetic factories. Burkart and Mayfield are experts in developing enzymatic pathways to synthesize a range of small molecules and macromolecules in photosynthetic organisms (polyketides, polyols, etc.). Enzymatic cascades will be stably integrated into genomes of algae and cyanobacteria by constructing genetic pathways for smal-molecule biosynthesis into inducible operons. Algae will be used when it is necessary for a product to be excreted from the cells to their surroundings, for instance in the case of therapeutic synthesis. Our aim for these materials is to be able to trigger a response of the living components to stimuli that will be encoded by genetic manipulation (i.e., we want to turn these materials on and off at will). We anticipate multiple methods of accomplishing this goal. For instance, J. Golden has previously described genetic logic gates, where an inducer molecule can turn on gene expression using riboswitches. J. Golden and S. Golden have demonstrated a NOT gate that turns genes OFF in response to a small molecule inducer. J. Golden’s group has made promoter devices with control of transcription and riboswitch devices that control translation; combinations of these regulatory devices can create an AND gate that produces gene expression (i.e., turns ON) only if two inputs are available. These logic gates will be used to drive biosynthesis to create responsive materials. Likewise, S. Golden is one of the world’s foremost experts on the circadian cycle and can control gene outputs in response to light or day/night cycles by using promoters from well-characterized genes. She has generated mutants that accumulate elevated pools of small metabolites by stopping the clock at a point in its cycle, and mutants that are more fit than the wild type when grown in a day/night cycle. Her expertise will be crucial to produce new materials that can synthesize and degrade a thermally insulating biofilm that is temporally controlled by a day/night cycle. Lastly, Pokorski has teamed with S. Golden to build matrices that support growth of cyanobacteria in 3D-printed parts.
These efforts in synthetic biology will be coupled to polymer synthesis and fabrication to create scaffolds that support sustained cell growth and/or activation of quiescent microbes. It is known from regenerative medicine that hydrated substrates (e.g., PEG or natural polymers like alginate, hyaluronic acid, etc), with relatively large pores, function best to ensure cell-growth. Preliminary studies have shown that cyanobacteria can be immobilized in alginate films for the production of hydrogen. It is less clear what would support the long-term growth of cyanobacteria, plant cells, algae or combinations thereof. Pokorski’s group will synthesize polymers that vary in charge, hydrophilicity/phobicity, and crystallinity, to develop a suite of polymeric substrates that can be swollen in aqueous conditions or could support desiccated cells that can be activated on demand. In more ambitious synthetic scaffolds, polymers will be synthesized to passively or actively release nutrients to minimize resource demand for materials over time. Lipomi and Pokorski will integrate the biologic components and polymers to yield new genetically encoded stimuli-responsive materials. Cell-laden polymer gels will be fabricated through direct-write 3D-printing or digital light processing (DLP) additive manufacturing to create complex geometric form factors. Genetically engineered bacteria can be patterned through soft-lithography (especially in desiccated or quiescent form), spin-coating or photolithography. It is anticipated that careful consideration of transport properties (Bae) for nutrient efflux and output and cell-material interfaces will need to be optimized for each individual microbe and desired gene products.
We illustrate with an example regarding chemical threat decontamination how unique expertise within IRG2 will be integrated to achieve scientific and societally relevant results that could not be achieved by any individual lab. J. Golden will engineer cyanobacteria with a riboswitch that turns ON in response to organophosphorous agents to produce acetylcholinesterase, a decontaminating enzyme. The engineered cyanobacteria will be coated onto a DLP-printed lattice scaffold of a water-swellable polymer, poly(hydroxyethyl methacrylate) (pHEMA) developed by Pokorski and Lipomi. The lattice framework will provide a higher surface area to volume ratio than a traditional membrane; this will encourage gas transport and enhance cell density over traditional monolithic materials. Initially, materials will be evaluated for biosynthetic output, enzymatic turnover, mechanical properties, and morphology. Bae will study and model transport phenomenon within this material to ensure influx of nutrients and efflux of waste products, primarily through control over the porosity of the materials. Effective diffusion coefficient of solutes in porous materials is mainly related to materials porosity (when the solute size is nearly constant and smaller than pore size), which can be easily manipulated through cross-link density or photo-cure time. Dutton will investigate effective methods to culture these engineered cyanobacteria at the polyHEMA interface, as she has done at natural material interfaces. Fluorescence imaging or nucleic acid detection-based methods will be employed for the measurement of organismal growth and the expression of target pathways. If polyHEMA is inappropriate for the chosen task, a wide swath of materials could be re-evaluated as scaffold materials including gelatin-methacrylate, poly(ethylene glycol), hyaluronic acid, and polyNIPAM, among others. Ultimately, we envision that this type of material will encourage cyanobacterial growth once placed in a humid environment (i.e., polyHEMA will swell to provide a source for water) and when the chemical threat (organophosphorous agent) is detected, the material will upregulate production of the decontamination enzyme. A technologically useful example of such a material would be an activatable filter or contact lens for first responders or military personnel.