The desire for space exploration of humans has been raised thousands of years ago and never faded. With the continuous improvement of astronautical technology, we finally have the chance to go beyond the Earth and possibly settle on other planets. As a neighboring planet, Mars is a reasonable first step in our exploration of space. To ensure the long-term survival and development of humans on Mars, a sustainable supply of nutrients is vital.

The sustainable supply of nutrients could be achieved in two existing ways: transportation from another place, and local plant production. On Mars or other alien planets, our home Earth is the only place we can transport supplies from. After research, we found that transporting from Earth faces plenty of problems such as long transport cycles, high costs, and high losses. Obviously, this way is far from feasible. But trying to grow crops on Mars to achieve local production seems not ideal, either. High soil toxicity would make plants difficult to survive and inedible; Low energy utilization and long harvest cycles would also result in extremely inefficient nutrient production.

Because of its easy culture, rapid production, low cost, and high energy utilization, maybe microbial production would be our best choice to produce nutrient production on Mars.

This year, ShanghaiTech_China developed an autotrophic sustainable multi-microbial production platform named Mini Bioproduction Cycle System (MBSC). We customized the platform for Martian conditions and launched the MBCS-Mars version. The MBCS-Mars is believed to be able to effectively use the atmospheric resources and light energy resources of Mars to continuously produce nutrients for human beings. To prove the concept, we performed a series of experiments and digital simulations to show the system could work. To get more details about how we prove our system, please go to our Prove of Concept page.

To achieve sustainable microbial production of nutrients on Mars, we need to overcome three obstacles. First, the system needs to use inorganic materials on Mars and convert them into organic matter effectively. Second, the artificial microbial "biosphere" must keep stable in the long term of Mars settlement. Lastly, we should come up with a practical production pattern for the MBCS to implement. To reach the above goals, we designed a ternary microbial symbiosis system, constructed the circulation and regulation of nutrients, and also proposed an innovative fermentation pattern as well as a photo-controlled and modular design.

Through investigation, we found that the atmosphere of Mars contains two elements mostly, carbon and nitrogen, mainly in the form of CO₂ and N₂. Carbon and nitrogen happen to be the two most important elements of life, and the vast majority of organic nutrients can be produced from these two elements. So we introduced three different types of microbes into our system. First, we introduced Synechococcus elongatus, a cyanobacterial species with the ability to fix CO₂ and produce sucrose through photosynthesis. In the past researches and iGEM projects, S. elongatus has been used as the autotrophic part of microbial symbiosis systems to provide carbon sources many times^{[1, 2]}. Second, we introduced Azotobacter caulinodans, which could convert N₂ into NH₄⁺ through nitrogenase to provide nitrogen source for the system. Finally, the production chassis Escherichia coli, or other fermentation chassis, can use sucrose and NH₄⁺ to produce more complex nutrients for human use.

We hope to build effective nutrient circulations among the above three microorganisms to attribute to their symbiosis. We found that NH₄⁺ is an ion that can be easily used by microorganisms, so there is no problem with the circulation of nitrogen sources. However, due to the preference of different microorganisms in using carbon sources, there are some problems in the circulation of carbon sources.

First, as told, S. elongatus can turn CO₂ into sucrose through photosynthesis. But wild-type S. elongatus fails to secrete their sucrose out of the cell to provide other cells with carbon sources due to its lack of natural sucrose transporters. Therefore, genetically modified sucrose-secretable S. elongatus was constructed by expressing the exogenous sucrose transporter gene cscB. Second, the wild-type E. coli K12 (like DH5α) strains have difficulties in using sucrose as the solo carbon source^[3]. So we transfected the β-glucosidase gene SacC into E. coli DH5α to enable the extracellular decomposition of sucrose into glucose and fructose, which could improve the ability of E. coli to utilize sucrose. Lastly, the A. caulinodans strain can only take organic acids as carbon sources, so it would not survive when sucrose was the only carbon source in the system^[4]. Luckily, we found that E. coli would excrete significant amounts of acetate (A. caulinodans can take acetate as its solo carbon source) when growing aerobically on glucose as the sole carbon source, which SacC-positive E. coli growing on sucrose will be in a similar situation^[5]. In this case, A. caulinodans could consume the unwanted by-product from E. coli and provide nitrogen sources for the system, which makes the system clever.

We found that previous attempts at artificial microbial symbiosis mostly failed because of the mismatched rates in the consumption and production of nutrients among the subpopulations^[1]. So, it is important to induce the regulation of nutrient by building intercellular genetic circuit feedback. We set these regulations between the production chassis E. coli and the nutrient suppliers (S. elongatus or A. caulinodans). Since E. coli receives nutrient for production, it is responsible for sensing the deficiency of nutrient and releasing intercellular signaling molecules to regulate nutrient suppliers.

When there is a deficiency of a certain nutrient (sucrose or NH₄⁺) in the system, nutrient receptor genetically engineered E. coli will release intercellular signaling molecules (we can also bacteria nutriental hormone, BNH) to increase the nutrient output cells nutrient output efficiency, to medium, add more nutrients to remove this lack of nutrient. To achieve the above transformation, we need three aspects of gene pathway design. They are the response to nutrient deficiency, the transmission of intercellular signals and the upregulations of nutrient export.

The response to nutrient deficiency relies on cellular intrinsic gene regulatory networks. When a lack of nutrient occurs in the cell, the expression of some metabolism-related genes increases under a series of network regulations^{[6, 7]}. By use of these related gene promoters(called starvation promoters) to guide the production of small signal molecular synthases, can the deficiency signal be converted into intercellular chemical signals, in which way the nutrient deficiency would be responded to. In MBCS-Mars, several endogenous carbon-starvation promoters and nitrogen-starvation promoters of E. coli were selected for characterization.

The transmission of intercellular signals in MBCS-Mars is implemented by quorum sensing. Here, we introduced the AHL family signaling system. AHL molecules can be synthesized by the expression of a single synthetase in the signal-sender E. coli. This small molecule is hydrophobic and can freely diffuse across the membrane. In the signal-receiver cell carrying the response element R-protein, the AHL molecules can bind to the R-protein and activate the expression of downstream genes^[8]. In the signal-sender E. coli. A homoserine lactone degrading enzyme gene was added to inactivate the AHL molecule at the end of the feedback phase. In the future, we also hope to characterize more intercellular signaling systems, so as to select the system with the most consistent signal response level and orthogonality.

The upregulations of nutrient export occur in carbon-supplier S. elongatus and nitrogen-supplier A. caulinodans. This kind of upregulations should be controllable and reversible, thus the system can be dynamically feedback. In S. elongatus, the sucrose transporter gene cscB is controlled by an inducible promoter to regulate the upregulation of sucrose export^[9]. In addition, controllable silencing of glycogen synthesis genes via sRNA was also designed to further enhance sucrose output, considering that microorganisms require higher sucrose content^{[10, 11]}. In A. caulinodans, the nitrogenase master regulator nifA was knocked out and then complemented by nifA controlled by inducible promoters to regulate the upregulation of nitrogen fixation rate^[12].

For implementation, suitable microbial fermentation equipment and pattern for MBCS-Mars is particularly important. Due to the fact that O₂ produced by the photosynthesis of S. elongatus is harmful to the nitrogenase of A. caulinodans, mixed liquid phase fermentation is not suitable for the system. So we proposed a novel fermentation model named Separate-Immobilized Fermentation. "Separate" means different engineered microorganisms ferment in separate fermenters, and "Immobilized" means microorganisms are immobilized into the medium as the stationary phase, and the culture fluid can flow between microorganisms as the mobile phase. This allows for independent production under optimum fermentation conditions while microorganisms maintain their nutrient exchange. Besides, we introduced hardware design including temperature control, flow rate control, gas control, and solution condition monitoring to the system. These digital controls of the fermentation equipment can further maintain the stability of the MBCS-Mars.

Modularization has been always considered a core idea in iGEM. In separate-immobilized fermentation, the immobilization may cause inconvenience to the harvest of the product and the replacement of microorganisms. Therefore, we proposed a photo-controlled plan to efficiently replace fermentation microorganisms. In the investigation, a reversible bacteria-material adhesion based on photocontrol protein has attracted our attention. The reversibility depends on a pair of light-controlled protein magnets, pMag and nMag, which exist as monomers in the dark and undergo rapid heterodimerization when exposed to 480nm blue light. The reversible adhesion can be achieved by displaying pMag on the surface of microorganisms and Reversible adhesion of engineered bacteria can be achieved by displaying pMag surface on engineered bacteria and modifying nMag on the surface of stationary-phase material under blue light control^[13]. Such photo-controlled reversible fixation is important because we want to save as much space, time, and production costs as possible on alien planets. This photo-controlled design allows us to efficiently replace microorganisms of different fermentation products in the same fermenter to save time and space. At the same time, the intracellular product can be harvested efficiently from the reaction vessel.

Although the core of our project is to provide a platform for microbial production, through genetic modification to increase the robustness of the microbial symbiosis system to meet long-term stability. In practical implementation, we have also proposed our final product form: through efficient photocontrolled reversible adhesion and membrane separation method to remove microorganisms, the fermentation broth containing nutrients were processed into nutritional canned food. In our partnership, we were also inspired by our partner BUCT-China that producing more delicious artificial cell meat should be another choice. To see more details, please go to the Implementation and Partnership page.

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