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We did our best to make our project useful and accessible to other iGEM teams. Read about:
Perchlorate reductase PcrAB reduces perchlorate to chlorite, the first step of complete perchlorate reduction pathway into chloride [1]. Using our pcrABD composite part [BBa_254G21JZ] we were the first to produce active perchlorate reductase in E. coli. This enzyme was never expressed actively outside of natively perchlorate reducing bacteria before. Due to the four naturally expressed proteins in perchlorate reductase operon pcrABCD, heterodimeric structure of the active enzyme complex and associated chaperone, neccessary for molybdopterin cofactor assembly, as well as the oxygen-sensitive Fe-S cluster cofactors, culturing and expression is highly demanding [2, 3]. We are very happy to share this result with future iGEM teams in search for a convenient way to produce active perchlorate reductase.
This lays the groundwork for other teams to further express, purify, characterize and engineer the enzyme as well as giving the standard biotechnology chassis E. coli the ability to fully detoxify perchlorate together with the following cld construct.
With this, we have investigated a low-cost photometric assay for testing the activity of perchlorate reductase. It can be performed without utilizing an anaerobic tent or expensive ion chromatography. Having verified our heterologus expression system, enabled us to be the first to achieve at least partial successful purification of the PcrAB complex using a Strep-tag II in a simple one step procedure [4, 5, 6]. This purification method is much more easily accessible than previously used methods to other iGEM teams, who desire to study perchlorate reductase in vitro, aim to make it less oxygen sensitive or otherwise improve its properties [3].
Chlorite dismutase (Cld) is the second key component of the perchlorate-reducing pathway, converting the produced toxic chlorite of the first step to harmless chloride and oxygen [1].
Using the his-tag technology, we purified Cld for characterization. Due to the extremely high turnover rates of this enzyme, characterization with standard methods is very challenging. To overcome this challenge, we optimized a low-cost protocol to measure the reaction rate of Cld using a photometer designed for completely different applications. This means the assay can hopefully be employed by every other iGEM team that has access to a standard photometer. We published the protocol in the part registry for any team to use in the future.
We have optimized an assay for the quantification of perchlorate reduction in in-vivo systems. This easy and rapid method can be used to measure the perchlorate concentration at low ppm levels. To perform it, only a chemical fume hood and a standard photometer are needed [7].
With this, future teams can tackle not just proving perchlorate reduction but thanks to the incredibly selective nature of the method, also use it to optimize bioremediation of perchlorate contaminations.
With the hardware we built, we not only gave a proof-of-concept for our designed low-cost, anaerobic bioreactor, but also contributed to open-source research by developing a Random Position Machine (RPM), a device that simulates microgravity on Earth.
The anaerobic bioreactor was designed to be inherently modular; in our current design we introduced temperature control as well as a pH sensor making it possible to efficiently and cheaply monitor culturing conditions. Building upon this, other iGEM teams could not only use the design as-is to culture other microorganisms anaerobically, they can easily improve the framework we provided for their needs: conveniently adding new functionality like in-line sampling for monitoring metabolites or protein production as well as liquid in/outputs for controlling pH- or nutrient conditions [8].
For best accessibility, our RPM was designed to be low-cost and 3D-printable. It is the first fully open source and verified RPM, and is even able to simulate microgravity in extended tests. We have shared all the 3D design files as well as assembly and calibration protocols in the hardware section, so any iGEM team, looking to examine the influence of extraterrestrial gravity on their chassis, now has the possibility to use it. With a total cost of roughly 150-180€, it is much cheaper than the commercially available alternatives that cost 8,000-50,000$ [9].
In conclusion we have contributed to open research and solving one of the major problems of colonizing Mars as best as we can. With the PcrAB and Cld characterization efforts we have provided easy to implement groundworks for the research of these enzymes. The perchlorate degradation assay provides the iGEM community with a way to efficiently monitor perchlorate detoxification in- and ex-vivo. With the RPM we have brought down the barrier for research on the effects of microgravity and last but not least we have developed a modular model-bioreactor to make larger scale cultivation of microorganisms easier and more accessible for other teams.
* 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 [11].
[1] N. Bardiya and J.-H. Bae, “Dissimilatory perchlorate reduction: a review,” Microbiological research, vol. 166, no. 4, pp. 237–254, 2011, doi: 10.1016/j.micres.2010.11.005.
[2] K. S. Bender, C. Shang, R. Chakraborty, S. M. Belchik, J. D. Coates, and L. A. Achenbach, “Identification, characterization, and classification of genes encoding perchlorate reductase,” Journal of bacteriology, vol. 187, no. 15, pp. 5090–5096, 2005, doi: 10.1128/JB.187.15.5090-5096.2005.
[3] S. W. Kengen, G. B. Rikken, W. R. Hagen, C. G. van Ginkel, and A. J. Stams, “Purification and characterization of (per)chlorate reductase from the chlorate-respiring strain GR-1,” Journal of bacteriology, vol. 181, no. 21, pp. 6706–6711, 1999, doi: 10.1128/JB.181.21.6706-6711.1999.
[4] D. L. Erbes and R. H. Burris, “The kinetics of methyl viologen oxidation and reduction by the hydrogenase from Clostridium pasteurianum,” Biochimica et biophysica acta, vol. 525, no. 1, pp. 45–54, 1978, doi: 10.1016/0005-2744(78)90198-5.
[5] M. Heinnickel, S. C. Smith, J. Koo, S. M. O'Connor, and J. D. Coates, “A bioassay for the detection of perchlorate in the ppb range,” Environmental science & technology, vol. 45, no. 7, pp. 2958–2964, 2011, doi: 10.1021/es103715f?
[6] M. D. Youngblut et al., “Perchlorate Reductase Is Distinguished by Active Site Aromatic Gate Residues,” The Journal of biological chemistry, vol. 291, no. 17, pp. 9190–9202, 2016, doi: 10.1074/jbc.M116.714618.
[7] I. Iwasaki, S. Utsumi, and C. Kang, “The Spectrophotometric Determination of Micro Amounts of Perchlorate by the Solvent-Extraction Method,” bull. Chem. Soc. Jpn., vol. 36, no. 3, pp. 325–331, 1963, doi: 10.1246/bcsj.36.325.
[8] C. Fung Shek, “Taking the pulse of bioprocesses: at-line and in-line monitoring of mammalian cell cultures,” Current opinion in biotechnology, vol. 71, pp. 191–197, 2021, doi: 10.1016/j.copbio.2021.08.007.
[9] D. Kim, Q. T. T. Nguyen, S. Lee, K.-M. Choi, E.-J. Lee, and J. Y. Park, “Customized small-sized clinostat using 3D printing and gas-permeable polydimethylsiloxane culture dish,” npj Microgravity, vol. 9, no. 1, p. 63, 2023, doi: 10.1038/s41526-023-00311-1
[10] D. C. de Geus, E. A. J. Thomassen, P.-L. Hagedoorn, N. S. Pannu, E. van Duijn, and J. P. Abrahams, “Crystal structure of chlorite dismutase, a detoxifying enzyme producing molecular oxygen,” Journal of molecular biology, vol. 387, no. 1, pp. 192-206, 2009, doi: 10.1016/j.jmb.2009.01.036
[11] E. C. Meng et al., “UCSF ChimeraX: Tools for structure building and analysis,” Protein science : a publication of the Protein Society, vol. 32, no. 11, e4792, 2023, doi: 10.1002/pro.4792.
