Why Synbio for Mars

Results We synthesized each of our constructs. However, because the constructs did not arrive until midway through August, we are still in the process of characterizing the transformants. Although our original concept was to detect DNA damage, the FRET mechanism has many other uses. For example, due to the number of different combinations of FRET chromophore pairs, a diversity of responses can be engineered into different reporting strains of bacteria to detect any signal that is mediated by a promotor. We have discovered this summer that microbial biosensors will be a necessary tool for synthetic biologists on Mars.

Project REGOBricks

1.  To map the larger perspective of applying our work in synthetic biology to space exploration, we interviewed a suite of distinguished scientists and administrators. Their wisdom is curated in a video series on our website.

2.  We publicized the potential of synthetic biology to a variety of audiences, including: 1.  Charlie Bolden, Stewart Brand, NASA Advisory Council 2.  Lunar Science Forum 3.  Maker Faire – we were awarded two Editor’s Choice Ribbons

3.  We led the largest collaboration between iGEM teams to date in the Jamboree working with teams across three continents to build:

Construct Our construct involves constitutive expression of CFP and YFP, bound to a C. thermocellum and C. cellulolyticum dockerin, respectively. To bring these two together, an inducible promoter produces a cohesin-cohesin fusion protein, one from each species.The cohesin fusion protein will bring together exactly one each of CFP and YFP to within 10 nm in order to generate a FRET signal.

Project FRETcetera

Biobricking a Biobrick The genetic cassette responsible for urease activity was curated using PCR from a recombinant E. coli species and added to the Registry as BBa_K656013. The gene in question (10.7 kb) was also sequenced for the first time in history, through a partnership with the Cornell Medical College. Ligation of our plasmid into backbone pBR322 and transformation of E. coli showed successful urease activity, observed through colorimetric changes on phenol-urea agar plates.

REGObricks investigates the potential of Sporosarcina pasteurii to catalyze biocementation for extraterrestrial regolith, setting the foundation for future space exploration. We showed proof of concept for biocementation’s potential to fuse analog extraterrestrial regolith, evaluated the space-worthiness of S. pasteurii, standardized the bacterium to current synthetic biology standards and modulated its useful urease function.

Characterization Biocementation trials of increasing scale were done with S. pasteurii, showing clear cementation at the microscopic level, and proof of concept at the macroscopic level. Bacteria were exposed to stratospheric conditions through stratospheric balloon flights, and assayed for their ability to withstand mechanical, barometric and ultraviolet stress. S. pasteurii samples secured on the surface of the carriage did not survive, though samples inside the carriage did show urease activity upon recovery, suggesting a future of this bacterium in the harsh vacuum of space.

Transformation Protoplast transformation of S. pasteurii was undertaken to investigate its potential as a future MICP chassis, with the hope of introducing additional functionality (antiobiotic selection, induced sporulation). Lysozyme was used to digest the cell wall, and DNA transformation induced with PEG. Cells were plated on sucrose-containing agar. Although we were not able to genetically transform S. pasteurii in time for the competition, we have tailored the process of protoplast transformation to better suit our organism and identified vitality of bacterium after digestion as a key function in future success.

Biocement Microbial Carbonate Precipitation (MICP) is a process in which organisms sequester carbon in crystals. Through the hydrolysis of urea, S. pasteurii is able to increase microenvironment pH, creating carbonate anion species to catalyze the precipitation of heavy metals.

Outreach

Abstract

The major advantage of a microbial sensor over inorganic sensors in the space environment is its ability to be mass produced from very little external input. Our goal is to create a novel mechanism of fluorescent biosensors that is more rapid than traditional sensors to serve as an early warning system on Mars. FRET Förster resonance energy transfer (FRET) is a phenomenon that occurs between two chromophores in close proximity (10 nm). If the emission spectra of one chromophore overlaps with the excitation of the other chromophore, then the first chromophore becomes a donor of energy to the second, instantly resulting in a greater emission of the second chromophore.

Cohesin-Dockerin Cohesin and dockerin are domains in the extracellular bacterial cellulosome and are species specific. One research group has created recombinant cohesin proteins which joins one cohesin each from C. thermocellum and C. cellulolyticum with an 11 residue linker, and a maximum end-to-end dimension of 98 +/-3 Å, or 9.8 nm. Because this distance is within the limit for FRET, we decided the hybrid cohesin protein can be the transcriptional product that brings together our two chromophores.

One of the critical challenges of space exploration is the limited payload mass that can be launched on a rocket and the difficulty of resupply mid-mission. Any long term settlement will require more resources than astronauts can initially bring with them. Synthetic Biology has the potential to revolutionize space exploration and settlement. Biological tools have a major advantage over classical tools: the ability to self-replicate and regenerate.

Problem in space travel Solution through synthetic biology

Payload is too costly to bring up to space Microbes are small and can be grown on site

Construction material too costly Project REGObricks and ISRU

Materials brought up non-renewable PowerCell uses solar energy to power cellular factories

Amplification Ligation Restriction

Sucrose Utilization Our tests suggest that it is possible to sustain E. coli growth on minimal media and sucrose. E. coli W has been used to produce bioplastics and nutrients, demonstrating the potential for PowerCell to support biological production systems.

On Mars it is highly impractical to transport growth media for microbes from Earth. PowerCell addresses this by generating sugar and nitrogenous products from the sun and Martian atmosphere, and secreting them for other organisms to use.

Anabaena PCC 7120 We selected our chassis for PowerCell based on several criteria: Carbon fixation: As a freshwater cyanobacteria, Anabaena photosynthesizes and natively produces sucrose as an osmotic balancer. Nitrogen fixation: Anabaena is diazotrophic, able to fix atmospheric nitrogen inside specialized cells called heterocysts. These heterocysts contain plasmodesmata that allow the exchange of nitrogenous products with other cells in the filament.

Construct We chose to focus on sugar secretion since there is evidence that nitrogenous products naturally leak from Anabaena.

CscB sucrose permease: a reversible sucrose transport protein from E. coli W Psac promoter: the 5’ UTR region of a photosystem I gene in Anabaena, exclusively expressed in photosynthesizing cells. This limits expression of CscB to vegetative cells. GFPmut3b: To verify correct expression and localization of our construct, detectable above background chlorophyll pigments

Transformation Conventional procedures do not work because Anabaena possesses restriction enzymes which digest foreign DNA. Our construct must be inserted with a helper plasmid which methylates the restriction site and a plasmid that enables bacterial conjugation.

Project PowerCell

iGEM’s first alumni network

Team: André Burnier1, Evan Clark2, Julius Ho1, Ryan Kent2, Lei Ma1, Eli Moss1, Jesse Palmer2, Max Song1, Jovian Yu1

(1) Brown University (2) Stanford University Advisors: Lynn J. Rothschild (Adjunct Professor, Molecular Biology, Cell Biology and Biochemistry, Brown University and Professor (Consulting), Human Biology, Stanford University), Gary Wessel (Professor of Biology, Molecular Biology, Cell Biology, & Biochemistry, Brown University)

Sponsors:

Scan for team wiki:

A Registry of Creative Human Practices

Team: Brown-Stanford