Synthetic biology can facilitate long-duration human spaceflight missions by enabling the production of nutrient-rich food from in-situ resources, by remediating waste, and by engineering synthetic probiotics to protect astronauts from microbes with increased virulence in microgravity. Such synthetic probiotics, which are an active area of research in the Tabor Lab, could also be engineered to produce and secrete molecules that strengthen the intestinal epithelium to help prevent radiation-induced inflammation of the digestive tract. Recent studies of microbial growth and gene expression suggest, however, that synthetic gene circuits may function unpredictably in space due to microgravity-induced changes in microbial physiology—the mechanisms of which are not well understood. Because synthetic gene circuits are highly sensitive to cellular physiological state, growth stage, and extracellular environment, both natural and synthetic gene regulatory networks must be characterized in space in order to evaluate performance changes resulting from microgravity, as well as to develop microgravity-insensitive organisms and circuits. This characterization requires the ability to precisely perturb cellular gene expression, but there is currently no platform available for dynamically controlling gene expression in space that enables this research. Microbial microgravity gene expression experiments to date have been limited to control by, at most, a single chemical inducer. When studying gene network dynamics, this strategy is confounded by the transport/diffusion delay of the chemical inducer across the cell membrane, effectively introducing a low pass filter upstream of the desired intracellular protein expression signal. This method is also not efficient for use in space-based applications because it requires complex and precise mixing fluidics, and is not amenable to multiplexing, as it requires separate reservoirs for each desired inducer molecule. Inducers are often highly sensitive to temperature and are generally unstable in aqueous solutions for long periods, and would likely not survive the (sometimes significant) pre-launch delay. A simple means to analyze gene expression dynamically and quantitatively in space is to use optogenetic systems, light-sensitive proteins that manipulate gene expression as well as other cellular processes including targeted protein degradation and dimerization. Towards this end, we recently demonstrated state-of-the-art quantitative gene circuit manipulation and evaluation using optogenetic methods to precisely control gene expression via multiple simultaneous channels. This “biological function generator” enables unprecedented analysis of the structure, state, and performance of gene circuits in any environmental context. Coupling these synthetic biological methods with the use of mathematical models to describe the relationship between input light and output promoter activity, we can precisely predict gene expression from two orthogonal optogenetic systems. We have also developed prototype systems for an optogenetic CubeSat platform capable of using light to study the effects of microgravity on gene expression and microbial physiology in space. Finally, we are in the process of engineering novel optogenetic light sensors with unique properties which will augment the optogenetic toolbox available to astrobiologists and synthetic biologists and improve the performance of those tools. As part of my NASA fellowship and in collaboration with my co-mentors, Dr. Tony Ricco & Dr. John Hogan, we have designed prototype hardware for a novel optogenetic CubeSat. Such a device would enable testing of synthetic gene circuit dynamics in space, thereby improving our understanding of how cellular physiology changes in response to microgravity. The hardware is based on related designs for ground-based optogenetic hardware developed with others in the Tabor Lab as well as CubeSat fluidic hardware designed by Dr. Ricco’s group at NASA Ames and which has flown on multiple missions. The general strategy has been to engineer the OptoGeneSat prototype on three fronts: hardware, wetware, and software. The hardware is the most tangible engineering product, comprising the optogenetic LED array and fluorescence & absorbance sensing arrays. The wetware engineering is focused on the 48-well PharmaSat-lineage fluidic system used to grow & dilute cells. We would like to make improvements to the PharmaSat card platform by enabling turbidostat-like dilution capability, and to optimize its geometry to improve well mixing for more uniform bacterial growth. The software front is designed to facilitate programming of the LED hardware to enable broader utility of this hardware platform for other NASA astrobiologists & synthetic biologists who may be less familiar with programming. Finally, we have also focused on light-sensor engineering and quantitative modeling to create improved light-sensing tools that can be precisely controlled with dynamic light inputs. We have applied a systematic dynamic characterization workflow, called frequency analysis, to create predictive models for our existing light-sensing two component systems (TCSs). We then adapted this to characterize improved versions of our light sensors with massively improved fold-inducibility. We have also developed a high throughput bioinformatic workflow that can identify putative new light sensors from sequenced genomes, a traditionally difficult task. We have isolated one such identified light sensor with a unique signaling architecture, and are currently characterizing its performance in E. coli in an effort to investigate whether it could display improved dynamic performance over our existing light sensors, such as robustness to noise, robustness to biological crosstalk, and robustness to copy number variation.