Project Abstract. . MicroMurals: Developing an educational tool that intersects synthetic biology and art. . MicroMurals utilizes synthetic biology to create bio-art, both as an educational and environmental tool. This was inspired by the need for synthetic biology to reach the public in an accessible and aesthetic way. MicroMurals provides an avenue to initiate discussions about synthetic biology with those at all levels of expertise, interest, and familiarity. Our team implemented an inducible hydrogel bio-ink system with biofilm-forming curli fibers fused to the chromoproteins aeBlue, asPink, and amajLime to create “microbial paints.” Hydrogels were harvested from E. coli and can be functionalized using VOC-uptaking cyclodextrin nanofibers or carbon-fixing microbes. We built a 3D bioprinter to print these hydrogels, as well as a handheld bioprinter for greater accessibility. Computer modeling was conducted for protein expression, protein folding, bioprinter extrusion, and bioreactor cell growth. Educational outreach events were held for multiple age groups, and BioArt exhibits were hosted on our campus to engage undergraduate students. Project Inspiration. . Our project was inspired by our discovery of a need for better educational tools to communicate synthetic biology. In our outreach events during last iGEM season, we noticed that it had been difficult to communicate this topic with younger audiences. In one event in particular at the Ithaca Sciencenter, we noticed that kids engaged more with the hands-on activity of creating collagen scaffolds using dry pasta and marshmallows. When it came to the verbal explanation of synthetic biology, even in the most basic terms, the kids were disinterested and kept looking toward the hands-on activities. This initially made our team realize that more creative methods would be needed to successfully get kids interested in synthetic biology and STEM. We soon realized that this applies to more than just young audiences. Much of the stigma surrounding synthetic biology could arise from hesitancy because people may not understand what the topic truly means. If you were trying to explain a synthetic biology journal article to someone who is not interested in or familiar with science, it would not get across to them successfully. However, if you shifted the method of communication to be more visual, there could be a greater level of interest and engagement. Our team thought that using synthetic biology to create art was an innovative method to make the field more intriguing, while also building on recent advances in the field of bio-art. We were inspired to develop this new application of synthetic biology as an educational tool to show how impressive synthetic biology can be, and the wonderful products that can come about through genetic engineering. We also hoped to use this tool to more easily open up a dialogue about the ethics and implications of synthetic biology and bio-art, to engage the public with both topics. To develop our project, we extended upon our research in hydrogel creation from our 2021 project. We looked through literature to find novel discoveries in the field of biomaterials, and decided to study bacterial-hydrogels in more depth [1]. Project Description. . Wet Lab Components. . To create our “microbial paints,” we have developed an autonomous system for bacterial secretion of fusion protein subunits that self-assemble to form a colorful hydrogel. Our constructs consisted of three different colored chromoproteins aeBlue (blue), asPink (pink), and amajLime (yellow) linked to the protein subunit csgA. This subunit is involved in curli biogenesis, which is the cellular process that develops the bacterial extracellular matrix [2]. As part of the curli biogenesis process, these fusion proteins were expressed within the csgAC operon alongside the csgEG operon. Figure 1 shows how the curli biogenesis system is used generally in other scientific literature. For our purposes, we wanted the fibers to be secreted into the extracellular environment, so we chose to remove the csgB gene in the csgBAC operon. Additionally, since csgF interacts with csgB by allowing it to detach from the cell surface when csgF is absent [4], we did not find the need to include csgF in our construct either. Though this gene allows for detachment, since we were concerned about construct size impacting cloning and transformation efficiency, we opted to keep our constructs as small as possible by only using the genes that were absolutely necessary for expression. We also based our fusion proteins on Duraj-Thatte et al. 2021, in which researchers were able to use fibrin-inspired knob and hole domains attached to the csgA protein fibers in order to allow for bacterial hydrogel self-assembly. Figure 2 shows the schematic from Duraj-Thatte et al. 2021 that shows the fibrin method of action in comparison to the knob and hole domain-linked proteins engineered in the paper. For our team, we incorporated chromoproteins as additional fusion proteins to the csgA-ɑ fibers. Figure 3 shows the constructs our team used for our project. Once these fusion proteins plus the gamma subunits are expressed after cell cultivation in the bioreactor our team designed, the protein monomers will be assembled, via chemical crosslinking, into a colorful hydrogel that can be used as printing ink (bio-ink) for a 3D bioprinter that we created, which comes with both a handheld and a stationary option. Genetic Design. . Starting With The Basics: Standard Parts. . The following parts are the basic components of our projects including the promoter, terminator, ribosome binding site and markers for our project. These parts are essentially the framework of our project. pSB1C3: A plasmid backbone with chloramphenicol resistance. BBa_K2042004: Inducible Promoter. BBa_B0014: Double Terminator. BBa_J61100: Ribosome Binding Site. Don’t Be Shy, Come On Out: Secretion System. . A frame needs a canvas! These parts help create the type VIII secretion system necessary to bring our art to life! This is done through curli nanofiber biosynthesis using the parts below CsgA-α (CsgA-Fibrin Knob Domain Fusion Protein): Stable Self-Assembling Monomer AND CsgA-γ (CsgA-Fibrin Hole Domain Fusion Protein): Stable Self-Assembling Monomer. . CsgA is the polymer forming unit of the curli biogenesis pathway which leads to biofilm formation in Escherichia coli. The wild-type csgA monomer can already self-assemble to form homopolymers outside of the cell. However, for our hydrogel art this interaction lacks sufficient stability. The mammalian protein fibrin arranges into fibrous chains to produce clots that hinder further blood loss. The polymerization of these chains relies on the interaction between N-terminal A- and B-knobs (alpha) and corresponding a- and b-holes (gamma) in the γ- and β-modules of fibrin [5]. When fused with protein csgA of the curli operon and chromoproteins, the fibrin knob domain (alpha) and the fibrin hole domain (gamma) will confer csgA the ability to stably crosslinking through the fibrin alpha and gamma domains. CsgC: Chaperone for secretion. csgC is a chaperone-like protein involved in the curli biogenesis pathway of gram-negative enterobacteria such as Escherichia coli. The curli biogenesis pathway results in the production of csgA nanofibers that allow bacteria to adhere to surfaces via biofilm formation. When the bacteria produces csgA, it is translocated to the periplasm in a Sec-dependent manner. There, csgC prevents the premature polymerization of csgA in the periplasm [6]. CsgE: forms secretion machinery with CsgG . csgE is an accessory protein in the Curli biogenesis pathway that allows for proper assembly of csgA subunits on the surface of gram-negative enterobacteria, such as Escherichia coli. Produced from the csgDEFG curli biogenesis regulatory operon, csgE localizes to the periplasmic space of E. coli. There, csgE acts as a chaperone protein by guiding the secretion of csgA through the outer membrane pore, CsgG, by forming a cap at the base of the pore[7]. By doing so, csgE can transport unfolded CsgA to CsgG for secretion [7]. CsgG: forms secretion machinery with CsgE. csgG is a key protein involved in the Curli biogenesis secretion-assembly pathway. As part of the csgDEFG operon, the csgG gene encodes for a peptide diffusion channel in the outer membrane of Escherichia coli [6]. Together with csgD, csgE, and csgF, csgG facilitates the proper secretion of curli subunits (i.e. csgA and csgB) from the periplasmic space. Once secreted, the csgA subunit (with the required assistance of csgB subunit) can aggregate and form amyloid fibrils at the cell surface. Let There Be Color: Chromoprotein Parts . . A canvas is boring without a little color! To bring our project to life, chromoproteins from iGEM’s 2011 Team Uppsala were utilized as the main form of bringing color to MicroMurals. Since the color is visible to the naked eye, they also serve as reporters for our plasmid expression. The following parts are the three chromoproteins we have chosen to use for our project. BBa_K864401: aeBlue, Blue Chromoprotein. BBa_K1033914: amajLime, Lime-Green Chromoprotein. BBa_K1033927: asPink, Pink chromoprotein. The csgA-γ fusion sequence contains the γ fibrin hole domain downstream of the csgA sequence while the csgA-α fusion sequence contains the α fibrin knob domain upstream of the csgA sequence. Because of the location of fibrin relative to csgA in CsgA-γ and CsgA-α, we have chosen to fuse these three chromoproteins to CsgA-α rather than to both CsgA-α and CsgA-γ monomers. Hardware. . In order for MicroMurals to be implemented into the real world, this hardware aspect is imperative in transforming the hydrogels into a product. For the hardware component of this project, we improved the bioreactor from Cornell iGEM’s 2021 Collatrix project, built a 3D bioprinter, and built a handheld bioprinter. For our constant volume continuous bioreactor, we have four components: the cultivation jar, base, feed, and electrical box. The cultivation jar is used to grow the bacteria by monitoring the pH, temperature, and O2 levels and supplementing with feed or base when necessary. This environment is agitated using a system of one propeller blade attached to a motor to keep the solution homogeneous. For the 3D printer, we developed a biocompatible printer by modifying the base of an old 3D printer (Makerbot Replicator 2), replacing the traditional 3D print head with a custom-designed syringe-based hydrogel extrusion head. The extrusion head uses a cartridge system, in which gel is preloaded into syringes and then pressure extruded out using a linear motor. This motor will rotate a 3D printed gear that will then move the syringe plunger down as printing occurs. For additional extrusion testing, we collected data on different hydrogels’ properties first: viscosity and compression testing. We also calculated the hydrogel’s resistance and fluid velocity when it flows through a cylindrical shape. This data generated a spectrum of hydrogel properties and to describe how these gels can behave while printing. We then ran tests on different extrusion methods, in which we used various shapes of syringes. The data provided a base for modeling the best extrusion method for our new 3D bioprinter. Furthermore, for our handheld bioprinter, our design consists of two cartridges/tubes for each color combined with a nozzle. This handheld bioprinter serves as a more accessible (and potentially cheaper in comparison to an actual 3D printer) option for artists or scientists looking to begin bioprinting. As for the software component of this project, we created an app that allows users to send in an image of a drawing to our 3D printer and then print this image using the bacteria produced hydrogel. The app contains a home page that acts similar to an Instagram or Facebook feed, in which users can see other images that have been printed. There is also a page which allows users to see what’s being printed at that exact moment, and there is a personal page to see updates on the images they have sent in. Human Practices. . Cornell iGEM focused closely on human practices related to bio-art this season, and looked into the intersection of art and science. We aimed to use educational programs, information graphics/flyers, and collaborative workspaces to bridge the gap between the worlds of art and science. Our interviews introduced us to aspiring artists, biology professors, bio-artists, ethics professors, and so many more people interested in the intersection of STEM and art. Discussions with such a variety of people offered us a chance to understand the technical and ideological background of the project on a deeper level. In addition to learning the technical details behind a hydrogel printer and chromoproteins, we found that the world of art is highly excited by the idea of incorporating scientific art into their work and pioneers have already begun to do so, whereas the science world lacks that enthusiasm. This realization inspired our educational, policy, ethics, and human practices work. We conducted programs, such as Splash!, that educated high school students on the women of synthetic biology in a hands-on manner. We lead a hands-on event at the Sciencenter, employing activities like Oobleck-making to model how our hydrogels will work. Furthermore, we had the pleasure to collaborate with a multitude of teams. The University of Rochester team visited Cornell for a lab tour, presentations, and to exchange materials for testing. Working with McGill and Concordia, we were able to host a hybrid mini jamboree to offer teams a chance to practice their presentations before the official jamboree. We also tried out our own bio-art skills by working with McGill, Queens, and Costa Rica to develop Biome, a bacterial picture book. MicroMurals is the next step in combining the creativity of making art with the technical aspects of biological engineering. Based on our research, it can also potentially incorporate the environmentally beneficial goals of carbon dioxide (CO2) and volatile organic compound (VOC) uptake. Researchers have already begun functionalizing these types of hydrogels for medical applications [1]. Inspired by this, our team researched ways to approach environmental applications of these colorful hydrogels to hopefully have our project impact the world on a greater scale. We figured that since the hydrogels were based on material that forms biofilms, we could re-embed bacteria into the hydrogels for programmable functions, for which proofs of concept do exist [8]. Further, we determined that adding additional nanofibers to our hydrogels would not only increase the structural integrity of them, but also potentially contribute to an environmental impact, such as through incorporating cyclodextrin (CD) nanofibers that have been shown to uptake volatile organic compounds (VOCs) [9]. Our vision for the future is that our bio-art could not only liven up any room, but could also filter the ambient air. We strive for our art to be accessible to individuals who are color blind, act as a carbon-friendly alternative to generic paints, and also educate and inspire the next generation of scientists and artists alike.