Overview. . Implementing MicroMurals by other users would mean implementing Cornell iGEM’s workflow for this project. Our workflow begins with strain cultivation, by growing E. coli that express our fusion protein subunits. Incubation of the colorful subunits with the csgA-γ producing strain allows for hydrogel self-assembly. Using methods from Duraj-Thatte et al. 2021, hydrogel filtration and collection can be performed. An additional component that may be incorporated depending on the application is also hydrogel functionalization for environmental applications. This is done through incorporation of other nanofiber materials such as cyclodextrin nanofibers, or other cellular components such as CO2-fixing bacteria. Once these hydrogels are ready, they can be loaded into our 3D bioprinting hardware components (tabletop 3D bioprinter and handheld 3D bioprinter). Once designs are uploaded either manually or through our app, they can be printed using our hardware and also live streamed through the app. Intended Use. . Our proposed end-users for MicroMurals are bio-artists and students. Bio-artists will be able to replicate our 3D printer using the hardware manuals we created (See Contribution), and begin using biomaterials to create various forms of art. Students can also interact with the tools we have in place, such as our app, to engage with the process. We envision that others will use our project to create art that will connect both the art and science worlds. We would implement our project into the real world by creating replicas of our 3D bioprinter and biomaterials for printing, as well as teaching others how to do this. These supplies could then be used by artists in their studios or scientists in their labs to create art that would eventually be showcased in museums or galleries. Some considerations we must make are the biomaterials with which we are printing. Currently, our team is using bacteria-made hydrogels, thus, we would need to ensure that this bacteria is dealt with for users to safely print. This is handled through the bacteria being lysed during the hydrogel processing protocol [1], thus no bacteria or bacterial remnants are present in the gel. Therefore, there is no biological risk from the bacteria that produce the gel itself. Additionally, we must take the disposal of any biomaterial waste into consideration. Though the hydrogels will not contain bacteria, the excess gel after printing may need to be disposed of through contained methods in case any bacterial contamination occurs. However, we do not expect this as the hydrogel would likely not have enough nutrients to sustain contaminating bacteria. Thus, standard trash disposal may also be an option. There are different considerations that must be made if hydrogel functionalization is performed. This functionalization step is most applicable to contained lab settings that are using the hydrogels for functional purposes. This is because one of the functionalization options is to embed microbes back into the hydrogel upon filtration. This can include embedding carbon dioxide-fixing microbes into the biofilm-based hydrogel, which is an environmental application of this project. In this case, a kill switch would be necessary. One option would be a kill switch that induces cell death in response to increased oxygen concentrations. This will be useful as diffusion of oxygen through a hydrogel decreases as more polymer chains are included within the gel [2,3]. Thus, functionalization would need to be done either using anaerobic microbes, or facultative anaerobes containing a plasmid that expresses a kill switch in the presence of elevated oxygen. Since the existence of such microbes that can survive in these gels while also cleaning the environment is not certain yet, our environmental approach using cyclodextrin nanofibers may serve to be more useful than functionalization through other microbes. Challenges. . Additional challenges to consider in regards to our 3D printer are limited bench/lab space, shelf stability, cost of materials, accuracy of printing, and housing the art. Many artists have expressed interest in creating bioart, however, it has been a challenge to find lab space to create in. It is imperative that we continue to make connections between artists and scientists with shared interest in bio-art as this field continues to expand. This can include providing training on the lab equipment and hosting workshops in which artists and scientists can learn more about how 3D printers and the printing software works. Furthermore, the shelf stability of our bacteria based printing is imperative to ensuring that our bacteria remains alive throughout the hydrogel production process. As 3D bioprinting becomes more popular, we must also consider the importance of using cost-effective materials. Most lab spaces have a limited amount of money that can be used to purchase supplies, and we must be willing to accommodate accordingly. For artists, the accuracy of the printing is imperative to creating the finely-detailed art that they envision. Lastly, housing the art is an additional challenge as it is not as simple as painting on canvas. If the art is exposed to air with no rehydration method, the art can take on new forms and change over time through dehydration that will naturally occur. Through interviews with artists, they seemed to be interested in bio-art as it often showcases art that changes over time. If users want to maintain a hydrogel material without these changes, the prints must be housed or enclosed somehow. Our team proposed permanently enclosed petri dishes as an option for this. Lastly, if the gels are microbe-functionalized, the art should be safely contained and used for the environmental application purpose rather than direct human use purpose. Since we are hoping to share this technology with the public, there is always a chance of dual use. As much as possible, we will seek to standardize and regulate the materials we give or show to bio-artists and scientists. We considered keeping some steps of the process proprietary, but this creates another challenge in terms of inaccessibility of the science behind this technology. However, this may be justifiable since it is meant for safety purposes and to avoid dual use concerns. Expansion. . We have also researched how we can take a business-based approach to expanding MicroMurals into a product. Business Model. . Our primary model is a direct-to-consumer tool with in-house production and distribution. Users will be able to submit designs and request a print using our MicroMurals app, which will then be conveyed to our in-house production center. The production team will then begin the process of converting the submitted images into a 3D printer compatible matrix, following which our biocompatible 3D printer produces the final printed art piece. The piece will then be stored, packaged, and shipped directly to the user. The next revenue stream involves partnerships with art galleries, universities, and schools. We hope to license our 3D-bioprinting technology to these larger organizations in order to allow them to produce their own forms of bio-art. Through this partnership, we will be shipping the 3D printer and all the complementary technology in order to allow these organizations to print art in-house. Once we successfully incorporate the CO2 uptake into the hydrogels, we hope to disrupt the print industry. By partnering with advertising firms to create large-scale signs, billboards and other print media using our vibrant, carbon-uptaking hydrogels, we are poised to gain a unique and large-scale physical presence by being a powerful solution for eco-conscious companies. Our handheld bioprinter has a unique business model, and is an extremely important component in making MicroMurals accessible to the public. The novel handheld bioprinter has numerous advantages including being extremely portable, hand-held, easy to use, inexpensive, and especially accessible. It also retains enough precision for accurate, good resolution, and high-quality prints. We hope to create MicroMurals “ink” cartridges, which would contain colorful hydrogels secreted by the bacteria as a complementary product to the handheld printer. In order to further our mission statement of inspiring people to pursue interdisciplinary research in STEM and the arts, we also hope to develop kits geared specifically towards students. These kits will include the handheld printer, a set of cartridges, and an interactive manual which will provide students with a simple overview of our project and synthetic biology. MicroMurals will be partnering with schools to supply these kits in bulk for a considerable discount. Partnered schools will have their own portal for the MicroMurals app, in order to have a private feed of work/designs submitted by their students open to feedback from faculty or collaboration with peers.