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A Random Positioning Machine (RPM) allows scientists to explore the mysteries of space right here on Earth by continuously reorienting samples along two axes, effectively “cancelling out” gravitational vector on the long run. By rapidly and uniformly changing orientation, it creates an environment where cells and particles behave as if they are in microgravity, opening a window into space biology without leaving the lab.
We successfully established an RPM system capable of reliably reaching microgravity.
These engineering cycles validated the core principle of our Random Positioning Machine (RPM): that a compact, modular platform can reproducibly generate stable low-gravity conditions in a laboratory setting. By addressing alignment tolerances and reducing friction in the sample holder, we achieved a system that overcomes oscillatory artifacts and maintains controlled gravity levels.
The ability to sustain effective microgravity without relying on parabolic flights, drop towers, or orbital experiments represents a significant breakthrough. It opens the door to continuous and cost-effective investigations of microgravity effects.
Thanks to its modular design, the RPM is highly adaptable to diverse research domains. Biological studies, such as cell culture, plant growth, or tissue engineering, can be combined with real-time imaging and environmental control. In material science, the platform enables research into crystallization, sedimentation, and fluid dynamics under reduced gravity. Furthermore, the system serves as an analogue testbed for planetary environments: by tuning the feed rate of the motors, researchers can simulate Martian (~0.38 g) or Lunar (~0.16 g) conditions, directly relevant to exploration missions and in-situ resource utilization (see Hardware).
The microbial enzyme perchlorate reductase (PcrAB) catalyzes the rate limiting step of the reduction of perchlorate into chlorite. Yet, the enzyme is highly complex, oxygen-sensitive, and its purification requires elaborate methods that are inaccessible to most laboratories, especially iGEM teams [2]. By developing accessible strategies for its expression and characterization, we are tackling a fundamental bottleneck in making perchlorate detoxification feasible for a broad range of applications.
To characterize perchlorate reductase we researched and established an enzymatic assay that works in cell lysate, because we were unsure about the feasibility of purifying perchlorate reductase aerobically, given the enzyme’s pronounced oxygen sensitivity. Consequently, we explored multiple strategies of characterizing perchlorate reductase and its more common related enzyme nitrate reductase directly in cell lysates.
Chlorite dismutase (Cld) catalyzes the second step of biological perchlorate reduction:
It rapidly decomposes chlorite into chloride and oxygen [6]
. As the enzyme’s biochemistry is
interesting not only for perchlorate degradation but also chlorite detoxification [7]
and oxygen
generation [8], we decided to purify and characterize Azospira oryzae GR-1 Cld in vitro.
To achieve this, a reliable and affordable activity assay is needed. As we cannot afford a Clark-type oxygen sensing
electrode, like done in most studies [9], we were happy to find an alternative
spectroscopic assay [10]. We tested this assay extensively and optimized it for our
application.
Through successive iterations of the DBTL-engineering-cycle we were able to substantially increase spectroscopic Cld activity assay’s reliability in our lab. We used it to easily evaluate total chlorite conversion as well as validate Cld’s kinetic parameters. All measurements were conducted on a standard photometer (Nanocolor UV/Vis II, Macherey-Nagel) designed for completely different applications (water/waste-water analysis). As photometers are standard equipment of many biological labs, we hope this assay is accessible to many other iGEM teams decreasing the need to invest into more accurate yet highly costly oxygen sensing electrodes.
Chlorite dismutase (Cld) is a well-characterized enzyme that is present in solution predominantly as a homopentamer, although its enzymatic activity is independent from oligomerization [14]. As suggested by the insight from Prof. Dr. Stein we investigated the feasibility of obtaining an active monomeric form of this protein. The ultimate objective of our design is to enable the fusion of monomeric chlorite dismutase (Cld) with perchlorate reductase (PcrAB), the enzyme responsible for the first step in perchlorate reduction. This fusion aims to produce a single, multifunctional protein capable of both reducing perchlorate and immediately detoxifying the resulting chlorite. This coupling would minimizes diffusion delays, thereby improving overall system efficiency [15].
While Cld’s enzymatic activity is independent of its oligomeric state, its native pentameric assembly poses practical challenges for fusion. Accommodating pentamerization in a fusion construct would require additional design constraints, such as linker optimization and interface stabilization. These complexities make a monomeric variant preferable, as it simplifies genetic fusion, improves predictability, and reduces the risk of misfolding or aggregation.
The objective of the cloning group is to design and assemble the perchlorate reduction operon pcrABCD and the cld gene in a plasmid vector and introduce it into Bacillus subtilis. Our work follows the Design–Build–Test–Learn (DBTL) engineering cycle to enable systematic and continuous improvement of our cloning strategies.
A central goal of our project is the development of a sensitive, reproducible and resource efficient detection system for microbial perchlorate degradation, since the perchlorate degradation ability of our engineered Bacillus subitilis needs to be verified. The usage of standard methods like ion chromatography were not feasible for us due to their high cost and different titrimetric method based on picric acid were rejected due to explosiveness, poor reproducibility, and safety risks [29]. This is why we opted and optimized a solvent extraction method with 1,2-dichloroethane (DCE) and methylenblue. With this method we were able to reliably quantify perchlorate at 0-5 ppm with a resolution of 0.1 ppm.
We successfully optimized a degradation assay and minimized the standard deviation to get better results
In the next steps, we will apply the methylene blue assay for the direct quantification of perchlorate degradation by our genetically modified bacterium. In addition, the low equipment and chemical requirements open up the possibility of developing the assay into a portable test kit, suitable for both terrestrial applications (e.g. environmental monitoring) and space missions (e.g., Mars exploration). Thus, our assay not only provides an essential contribution to the validation of our chassis, but also represents a scalable, safe, and reproducible method for research and applications beyond iGEM. While detection in the ppb range (e.g. 5 ppb) would be desirable to measure the needed concentration for drinkable water, this is not achievable with the methods available to us.
* Molecular graphics and analyses performed with UCSF ChimeraX, developed by the Resource for Biocomputing, Visualization, and Informatics at the University of California, San Francisco, with support from National Institutes of Health R01-GM129325 and the Office of Cyber Infrastructure and Computational Biology, National Institute of Allergy and Infectious Diseases [16].
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