iGEM 2025 — Team tudarmstadt


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![wengineering success illustration](https://static.igem.wiki/teams/5634/wiki/astroowl/engineur.svg)

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**Read our engineering efforts for:**

* Design and building of Random Positioning Machine
* Expression and characterization of perchlorate reductase in *E. coli*
* Optimization of chlorite dismutase activity assay
* Engineering of monomeric chlorite dismutase mutants
* *B. subtilis* engineering with chlorite dismutase
* Optimization of perchlorate quantification assay

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## Random positioning machine

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.

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### Summary

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](hardware#rpm)).

Next

## *In vitro* characterization of perchlorate reductase and nitrate reductase

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.

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## Chlorite dismutase activity assay

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.

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### Summary

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 monomerization

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](human-practices#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.

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## Engineering of *B. subtilis*

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.

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## Detection method for the microbial degradation of perchlorate

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.

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### Summary

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](https://2025.igem.wiki/tudarmstadt/results#degradation-result), 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].

## References

[1] A. M. Dias, L. Wenyan, and I. Tesia, “Investigation into Space Effects on Biofilm Growth Using Simulated Microgravity,” 2022. [Online]. Available: https://ntrs.nasa.gov/api/citations/20220003678/downloads/ICES\_215.pdf

[2] Matthew D. Youngblut, “Perchlorate Reductase Is Distinguished by Active Site Aromatic Gate Residues,” 2016. [Online]. Available: https://doi.org/10.1074/jbc.M116.714618
[3] 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.

[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] T. G. M. Schmidt and A. Skerra, “The Strep-tag system for one-step purification and high-affinity detection or capturing of proteins,” Nature protocols, vol. 2, no. 6, pp. 1528–1535, 2007, doi: 10.1038/nprot.2007.209.

[6] I. Schaffner, S. Hofbauer, M. Krutzler, K. F. Pirker, P. G. Furtmüller, and C. Obinger, “Mechanism of chlorite degradation to chloride and dioxygen by the enzyme chlorite dismutase,” Archives of biochemistry and biophysics, vol. 574, pp. 18–26, 2015, doi: 10.1016/j.abb.2015.02.031.
[7] C. G. van Ginkel, G. B. Rikken, A. G. Kroon, and S. W. Kengen, “Purification and characterization of chlorite dismutase: a novel oxygen-generating enzyme,” Archives of microbiology, vol. 166, no. 5, pp. 321–326, 1996, doi: 10.1007/s002030050390.

[8] A. L. Markhard, J. G. McCoy, T.-L. To, and V. K. Mootha, “A genetically encoded system for oxygen generation in living cells,” Proceedings of the National Academy of Sciences of the United States of America, vol. 119, no. 43, e2207955119, 2022, doi: 10.1073/pnas.2207955119.
[9] van Ginkel, C., Rikken, G., Kroon, A. et al., “Purification and characterization of chlorite dismutase: a novel oxygen-generating enzyme.,” Arch Microbiol, no. 166, 321–326, (1996)., doi: 10.1007/s002030050390.

[10] M. Philippi, H. S. dos Santos, A. O. Martins, C. M. N. Azevedo, and M. Pires, “Alternative spectrophotometric method for standardization of chlorite aqueous solutions,” Analytica chimica acta, vol. 585, no. 2, pp. 361–365, 2007, doi: 10.1016/j.aca.2006.12.053.

[11] D. M. Freire et al., “The homopentameric chlorite dismutase from Magnetospirillum sp,” Journal of inorganic biochemistry, vol. 151, pp. 1–9, 2015, doi: 10.1016/j.jinorgbio.2015.07.006.

[12] Stefan Hofbauer Clemens Gruber, Katharina F Pirker, Axel Sündermann, Irene Schaffner, Christa Jakopitsch, Chris Oostenbrink, Paul G Furtmüller, Christian Obinger, “Transiently produced hypochlorite is responsible for the irreversible inhibition of chlorite dismutase,” 2014, doi: 10.1021/bi500401k.

[13] M. Mesa, J. A. Pereañez, L. M. Preciado, and C. Bernal, “How the Triton X-100 modulates the activity/stability of the Thermomyces lanuginose lipase: Insights from experimental and molecular docking approaches,” International journal of biological macromolecules, vol. 120, Pt B, pp. 2410–2417, 2018, doi: 10.1016/j.ijbiomac.2018.09.009.

[14] 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.

[15] H. Ueno, K. Oishi, Y. Sayato, and K. Nakamuro, “Oxidative cell damage in Kat-sod assay of oxyhalides as inorganic disinfection by-products and their occurrence by ozonation,” Archives of environmental contamination and toxicology, vol. 38, no. 1, pp. 1–6, 2000, doi: 10.1007/s002449910001.

[16] 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.

[17] E. Krissinel and K. Henrick, “Inference of macromolecular assemblies from crystalline state,” Journal of molecular biology, vol. 372, no. 3, pp. 774–797, 2007, doi: 10.1016/j.jmb.2007.05.022.

[18] J. Jumper et al., “Highly accurate protein structure prediction with AlphaFold,” Nature, vol. 596, no. 7873, pp. 583–589, 2021, doi: 10.1038/s41586-021-03819-2.

[19] Y. Zhang and J. Skolnick, “TM-align: a protein structure alignment algorithm based on the TM-score,” Nucleic Acids Research, vol. 33, no. 7, pp. 2302–2309, 2005, doi: 10.1093/nar/gki524.

[20] Zhang Lab, TM-align. [Online]. Available: https://zhanggroup.org/TM-align/TMalign.cpp (accessed: Jul. 29 2025).

[21] Y. Liu, X. Yang, J. Gan, S. Chen, Z.-X. Xiao, and Y. Cao, “CB-Dock2: improved protein-ligand blind docking by integrating cavity detection, docking and homologous template fitting,” Nucleic Acids Research, vol. 50, W1, W159-W164, 2022, doi: 10.1093/nar/gkac394.

[22] Y. Liu, L. Xie, and H. Li, CB-Dock2 Web Server. [Online]. Available: https://cadd.labshare.cn/cb-dock2/php/index.php (accessed: Jul. 29 2025).

[23] U. K. Laemmli, “Cleavage of structural proteins during the assembly of the head of bacteriophage T4,” Nature, vol. 227, no. 5259, pp. 680–685, 1970, doi: 10.1038/227680a0.

[24] A. Grote et al., “JCat: a novel tool to adapt codon usage of a target gene to its potential expression host,” Nucleic Acids Research, vol. 33, Web Server issue, W526-31, 2005, doi: 10.1093/nar/gki376.

[25] Kazusa DNA Research Institute, Codon usage table.

[26] Y. Xia, K. Li, J. Li, T. Wang, L. Gu, and L. Xun, “T5 exonuclease-dependent assembly offers a low-cost method for efficient cloning and site-directed mutagenesis,” Nucleic Acids Research, vol. 47, no. 3, e15, 2019, doi: 10.1093/nar/gky1169.

[27] D. G. Gibson, L. Young, R.-Y. Chuang, J. C. Venter, C. A. Hutchison, and H. O. Smith, “Enzymatic assembly of DNA molecules up to several hundred kilobases,” Nature methods, vol. 6, no. 5, pp. 343–345, 2009, doi: 10.1038/nmeth.1318.

[28] J. S. Sharp and D. H. Bechhofer, “Effect of translational signals on mRNA decay in Bacillus subtilis,” Journal of bacteriology, vol. 185, no. 18, pp. 5372–5379, 2003, doi: 10.1128/JB.185.18.5372-5379.2003.

[29] A. Bolliger, “Die titrimetrische Bestimmung von Perchloraten Ein Beitrag zur Mikroanalyse des Kaliums,” (in De;de), Fresenius, Zeitschrift f. anal. Chemie, vol. 94, 11-12, pp. 403–408, 1933, doi: 10.1007/BF01352723.

[30] 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.

[31] T. Ali-Vehmas, M. Louhi, and M. Sandholm, “Automation of the resazurin reduction test using fluorometry of microtitration trays,” Zentralblatt fur Veterinarmedizin. Reihe B. Journal of veterinary medicine. Series B, vol. 38, no. 5, pp. 358–372, 1991, doi: 10.1111/j.1439-0450.1991.tb00883.x.

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{% block title %}Results{% endblock %}
{% block lead %}**Our achievements:**

* Verification of Random Positioning Machine
* Active expression of perchlorate reductase in *E. coli*
* Characterization of chlorite dismutase *in vitro*
* Analysis of chlorite dismutase mutants
* Evaluation of engineered *B. subtilis*
* Validation of a perchlorate degradation assay *in vivo*

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![results presenter illustration](https://static.igem.wiki/teams/5634/wiki/astroowl/resultpresenter.svg)

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## Random positioning machine results

#### Key Insight

Diffusion of probes rotating in the RPM took approximately **twice as long** compared to static controls.
This proves the RPM's ability to **reduce diffusion rates** by simulating microgravity conditions.

**Goal:** The aim of this project was to create a validated, open-source Random Positioning Machine (RPM) that allows researchers and students to simulate microgravity and partial gravity in a laboratory setting. Commercial RPMs are prohibitively expensive, preventing many groups from investigating how biology behaves in space-like environments.

We designed and validated a low-cost, open-source Random Positioning Machine as a microgravity simulator for just 150-180 €. Unlike existing DIY projects, our RPM has an operating system and was experimentally verified using a mobile phone IMU (LSM6DSOX). At a feed rate of 50 mm/min the averaged effective gravity reached 0.028-0.070 *g* (see Table 1), confirming simulated microgravity. By adjusting the feed rate, any gravitational acceleration could be simulated at the cost of more centrifugal force.

##### Goal

Create a validated, open-source RPM for simulating microgravity/partial gravity.

##### Method

Feed-rate control + IMU validation; dye diffusion test inspired by NASA’s protocol.

##### Result

Effective gravity down to **0.028-0.070 *g*** at 50 mm/min; ~2× slower diffusion under rotation.

Table 1: Measured average effective gravity at different feed rates during rotation.
Measurements at 100 and 50 mm/min were 10 min; other points were 1 min starting after 3 s.

| F: Feed rate [mm/min] | Average effective gravity (× *g*) |
| --- | --- |
| 400 | 0.73 |
| 300 | 0.52 |
| 200 | 0.28 |
| 100 | 0.09 |
| 50 | 0.053 |

To validate the RPM’s ability to simulate microgravity, we developed and optimized a simple dye diffusion test inspired by NASA’s protocol [1]. Using this method,
we demonstrated that the rotating sample required roughly twice as long to fully diffuse as the static control, clearly showing that the RPM effectively reduces diffusion and mimics microgravity conditions.

### A visual detection: Dye diffusion test

Inspired by NASA’s dye diffusion test
[1],
we optimized and changed the protocol to observe the dye diffusion easily at home. With 0.5 % agar and 50 ml Falcon tubes it was possible to distinguish between a static and rotating sample (see Figure 1). After 20 minutes, the dye in the static tube had already started to spread evenly, while it remained concentrated at the center in the RPM tube. After 50 minutes, the static tube was fully diffused, whereas the RPM tube still displayed a distinct undyed region. Only after 100 minutes did the RPM tube appear uniformly colored.
In blind evaluations, a handful of team members were consistently able to recognize the RPM samples, confirming the robustness of the effect. The final protocol is inexpensive, easy to replicate, and can even be reproduced at home using food coloring and a pipette.

![Dye diffusion test comparing a static control (left) vs. rotating RPM tube (right)](https://static.igem.wiki/teams/5634/wiki/engineering/rpm/dyediffusiontest.svg)

**Figure 1:** Dye diffusion test of a static control on the left of every picture and a rotating on the
right in 0,5% Agar and a feed rate of 50 mm/min

The RPM can be used for future *in vivo* research studies, particularly in combination with our water purification system. For example, it allows controlled studies of plant growth with purified water under Martian gravity on Earth.

## Perchlorate reductase characterization

#### Overview

We set out to make the complex, oxygen-sensitive enzyme perchlorate reductase (PcrAB) accessible for iGEM-level expression and characterization.
Through four [engineering cycles](https://2025.igem.wiki/tudarmstadt/engineering#perchlorate-reductase) we designed, built, and tested expression constructs and began rational enzyme engineering to improve activity.
Below we summarize key experimental outcomes, their significance, and next steps.

### PMS/NADH Lysate Assay

We aimed to express active PcrAB with a Strep-Tag II for straightforward purification. We successfully cloned a
*pcrABD* operon lacking *pcrC* into an IPTG-inducible expression system and expressed it in *E. coli* BL21(DE3).
Slow growth under anaerobic conditions was observed, but lysates showed perchlorate-reducing activity (see Figure 2).
Using phenazine-methosulfate (PMS) and NADH, we measured NADH oxidation corresponding to perchlorate reduction at 340 nm in cuvettes overlaid with mineral oil
[2]. Lysate from cultures induced with 100 µM IPTG showed the highest activity, giving a linear NADH consumption rate of ~2.7 nM min⁻¹, while GFP expressing negative controls showed no activity (see Figure 2,3).
Compared to cultures induced with lower concentrations, such as 50 µM, this condition yielded the strongest activity, as depicted in Figure 3.

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### Purification of Strep-Tagged PcrAB

Next we sought to easily purify PcrAB by utilizing its Strep-Tag II for affinity chromatography. Purification yielded two bands on the SDS-PAGE for the wash fraction, suggesting the enzyme bound too weakly to the resin (see Figure 4). Nevertheless, thereby obtained PcrAB seems to be pure.
PcrA exhibits mass of 104 kDa and PcrB 37 kDa
[3].

![SDS-PAGE of purified PcrAB](https://static.igem.wiki/teams/5634/registry/pcrab/sds-gel-purification2.avif)

**Figure 4:** SDS-gel of PcrAB purification. The before/after induction and
cell lysate analysis was performed on a separate gel ([original data](https://static.igem.wiki/teams/5634/registry/pcrab/sds-gel-sdm-cell-lysat.webp))
than the purification process ([original data](https://static.igem.wiki/teams/5634/registry/pcrab/2-purification-last-two-lanes.webp)).

### Site directed mutagenesis of *pcrA*

Plasmids carrying verified *pcrA* Y165H and W461H mutations were transformed into *E. coli* BL21(DE3) for small-scale expression under the same anaerobic and induction conditions as our wild-type *pcrABD* construct.
Lysates were evaluated by the PMS/NADH assay to compare NADH consumption rates, see Figure 5.

**Figure 5:** Activity measurements of cell lysates expressing the *pcrA* Y165H and W461H mutations compared against buffer-only control.

Assays of the Y165H and W461H mutations showed no detectable perchlorate degradation compared to the Tris-HCl buffer control, indicating that the enzyme was not functional.
These histidine substitutions likely disrupted the critical aromatic “gate” residues of PcrA, impairing substrate binding and electron transfer [3].
Alternatively, oxygen sensitivity of the employed assay obscured activity.

#### Conclusion

Our work demonstrates, for the first time, functional expression of active, affinity-tagged perchlorate reductase in *E. coli* and establishes an assay platform for its characterization. These advances lower the technical barrier for studying and engineering this key enzyme, moving iGEM and the broader scientific community closer to deployable microbial solutions for perchlorate detoxification on Earth and Mars.

## Chlorite dismutase *in vitro* characterization

Chlorite dismutase (Cld) catalyzes the rapid detoxification of chlorite into chloride and oxygen, see Figure 6
[4]. This is the second step of biological perchlorate reduction
[5].

![Cld reaction: chlorite to chloride + oxygen](https://static.igem.wiki/teams/5634/wiki/results/cld-reaction-white.webp)

**Figure 6:** Cld catalyzes the transformation of chlorite into chloride and oxygen.

As Cld is critical to break down toxic chlorite to prevent it from harming our chassis
[5], we decided to purify and characterize *Azospira oryzae* GR-1 Cld extensively *in vitro* and derive implications for its implementation in *Bacillus subtilis*.
We were able to successfully purify it via IMAC and investigate an alternative, low-cost and fast activity assay.
Details about methodology can be read at the
[iGEM part registry](https://registry.igem.org/parts/bba-25vz3x3w).

### Production and purification

Cld was produced in *E. coli* BL21(DE3) pLysS and purified using a HisTrap column (Cytiva)
([read details](https://registry.igem.org/parts/bba-25vz3x3w)).
The process was analyzed on an SDS-PAGE (Figure 7). Overproduction upon induction as well as binding and elution
from the HisTrap column were verified. Cld stock is very pure, with only slight contamination by a ~60 kDa protein.

Protein (monomer) concentration was determined to (63 ± 7) µM via Bradford Assay
[6].
Yield was probably reduced due to poor elution from the column, in contrast to descriptions in the literature
[7].
Nevertheless, obtained Cld stock was completely sufficient, as only nM concentrations are needed for activity assays.

![Cld purification SDS-PAGE](https://static.igem.wiki/teams/5634/wiki/results/cld-purification-white.webp)

**Figure 7:** SDS-PAGE analysis confirmed successful Cld purification.

### Heme Quantification

#### Pyridine hemochrome assay

To quantify heme *b* amount in Cld stock solution, the pyridine hemochrome assay was employed. This assay exploits the fact that pyridine forms complexes with heme groups,
which show distinct absorption spectra upon reduction to the ferrous state by sodium dithionite. From absorption at peak subsequently heme concentrations can be calculated using the extinction coefficient ε556nm=34.7 mM-1cm-1
[8].
This assay was used to verify if every Cld monomer is bound to a prosthetic heme-group, as in that case Cld and heme concentrations should be equal. Any free heme should have been removed during the two chromatographic steps during purification. In Figure 8 absorption spectra of oxidized and reduced heme from purified Cld are shown.

**Figure 8:** Absorption spectra of pyridine hemichrome (oxidized Fe) and pyridine hemochrome (reduced Fe) from purified Cld (1:5 dilution).

Heme concentration could be determined to 68 µM. This value is within error range of (63±7) µM Cld protein concentration. Thus heme and Cld concentrations can be considered equal within (substantial) measurement inaccuracies and so every Cld subunit should have a prosthetic heme-group bound. Because tight heme binding is only possible with a correctly folded ferredoxin-like binding pocket [5], every Cld molecule should be correctly folded and active.

### Catalytic activity

Degradation of chlorite into chloride and oxygen was monitored spectroscopically at 260 nm
[9,
 10]. This method allows fast and easy measurement of chlorite concentrations.
As neither of the products absorbs light at 260 nm, direct evaluation of leftover chlorite concentration is possible
[11].

#### Read details about activity assay

Because chlorite degradation by Cld is very fast, it is critical to start the measurement immediately
after adding Cld to chlorite solution. To ensure efficient mixing nevertheless, always 200 µl Cld
solution was pipetted to 200 µl chlorite solution. Control experiments with 200 µl
heat-inactivated Cld show stable absorbance, see Figure 9a, indicating both solutions get thoroughly
mixed through flux caused by pipetting. Otherwise, diffusion of chlorite/buffer into the light path should
have caused fluctuations in absorbance.

By applying this procedure, kinetics could be evaluated on a Nanocolor UV/Vis II spectrometer designed for
completely different applications (water/waste-water analysis). As most labs have access to standard
photometers, we hope this assay is accessible to many iGEM teams.

#### Enzyme inactivation by hypochlorite

Our results show that Cld gets inactivated after a certain amount of turnovers
and thus does not decompose chlorite completely.
Cooler reaction temperatures lead to higher turnover numbers.

#### Read about temperature effects on Cld inactivation

As can be seen in Figure 9a, at room temperature absorption stabilizes around 0.11, indicating not all
chlorite gets degraded. This is because hypochlorite is produced as intermediate during chlorite degradation,
and it probably reacts with the heme-group or tyrosine residues leading to irreversible Cld inactivation
[10].
Plotting the chlorite turnover number within 1 min against initial chlorite concentration reveals enzyme
inactivation after conversion of ~0.3 µmol chlorite per unit of Cld (Figure 9b), similar to previously
reported data (0.38 µmol/U for Cld from *Dechloromonas agitata* CKB
[4]).

Comparison of total chlorite conversion at room temperature and when mixing ice-cold substrate and Cld solutions
together showed significantly more chlorite converted for the latter (p<0.1% for Figure 9a after 1 min),
see Figure 9. This temperature influence on Cld inactivation is known
[11]
and might be explained by the fact
that transiently produced hypochlorite needs to escape the active site in order to inactivate Cld
[10].
As lower temperature slows down diffusion, this escape might become less likely.

**Figure 9a:** Time traces of 1 mM chlorite degradation by 6.3 nM Cld, monitored spectroscopically at 260 nm. Cld preheated to 90°C for 5 min serves as negative control **(red)**. Mixing Cld and chlorite solutions together ice-cold and starting measurement immediately in room-temperature spectrometer results in higher total chlorite conversion (**blue**, n=4) than mixing room-temperature solutions (**orange**, n=5). (y-intercept of curves was chosen manually.)

![Cld inactivation turnover (Figure 9b)](https://static.igem.wiki/teams/5634/wiki/results/cld-inactivation-white-1.webp)

**Figure 9b:** Chlorite converted within 1 min per unit of Cld in dependence of initial chlorite concentration shows that Cld is inactivated after a finite number of turnovers. This turnover number can be increased by mixing solutions together ice-cold (both n=3).

Despite changing total chlorite conversion, reaction temperature did not cause significant activity changes. For Figure 9a initial velocities were (11.6±1.5) µM/s and (10.2±1.4) µM/s for ice-cold and room temperature reactions respectively (p>24%).
Thus, lower reaction temperature can be used to improve activity assay accuracy, by increasing initial linear phase of reaction.

#### Spontaneous activity decay on ice

During some recorded kinetics, poor linearities in Lineweaver-Burk-Plots were obtained. This could be attributed to the fact that Cld activity decays when stored on ice for a measurement relevant time scale. Activity only decays in low concentrated solutions.

#### Read details

In Figure 10 activity remaining after storing different Cld solutions on ice is displayed.
Complementary to activity, also total chlorite conversion was used to evaluate decrease of active Cld.

![](https://static.igem.wiki/teams/5634/wiki/results/cld-decay-white.webp)

**Figure 10:** Activity and total chlorite conversion (within 2 min) in dependence of incubation time of Cld on ice. Cld was incubated in 6.3
nM solution, 630 nM solution and 6.3 nM solution with 0.001% Triton X-100 (AppliChem).
Reaction was started ice-cold with 1 mM chlorite and 3.2 nM Cld. For better visualization, freshly prepared Cld’s values without Triton were set to 100%. Technical triplicates (n=3) were evaluated.

Storing 6.3 nM Cld solution (typical assay concentration) for 100 min on ice leads to ~30%
activity/chlorite conversion loss (p<5%). This substantially tampers kinetic measurement series.
In contrast, both 630 nM Cld solution as well as 6.3 nM Cld with 0.001% Triton X-100 did not
exhibit significant activity/chlorite conversion loss (all p>10%).

The observed decay of active enzyme is surprising, as pentameric Cld is known for its thermostability
[12].
The instability of less concentrated Cld solutions could be due to a shift in the oligomerization equilibrium
toward monomeric Cld. Monomeric Cld is expected to be less stable than pentameric due to exposed hydrophobic
surfaces which are covered in pentameric form.

Triton X-100 could accordingly stabilize pentameric or monomeric Cld, as it stabilizes other enzymes by
masking exposed hydrophobic surfaces
[13]. Thus, Triton might be suited to stabilize low-concentrated Cld
in order to perform accurate assays. However, further experiments would need to verify that activity really
does not decay in presence of Triton first, as measurements shown here exhibit high standard deviations
which might obscure significant activity loss.

#### Michaelis-Menten kinetic

Previous findings were used to conduct kinetic measurement series: Solutions were mixed ice-cold to prolong initial linear reaction phase and 6.3 nM Cld solution was prepared freshly for each measurement from 630 nM stock. Initial velocities were obtained by linear or exponential regression; an example is shown in Figure 11a. Lineweaver-Burk-Plots are shown in Figure 11b, derived kinetic parameters are listed in Table 2.

![Exemplary degradation curve of 1 mM chlorite by 3.2 nM Cld](https://static.igem.wiki/teams/5634/wiki/results/cld-curve-fit-white.webp)

![Lineweaver–Burk plot for 3.2 nM Cld (n=3)](https://static.igem.wiki/teams/5634/wiki/results/cld-lineweaver-white.webp)

**Figure 11:**
**(a)** Exemplary degradation curve of 1 mM chlorite by 3.2 nM Cld. Linear and exponential fit were conducted in Microsoft Excel.
**(b)** Lineweaver–Burk plot for 3.2 nM Cld (n=3). Linear regressions are shown, both for initial velocities obtained by linear as well as exponential regression.

Table 2: Comparison of kinetic Michaelis-Menten parameters using a linear or exponential fit and literature values
[14]
.

| Parameter | Linear fit | Exponential fit | Literature |
| --- | --- | --- | --- |
| kcat [1/s] | 2800 ± 410 | 4910 ± 750 | 1170 |
| KM [µM] | 178 ± 26 | 377 ± 47 | 170 |

#### Read discussion

Both Lineweaver-Burk plots exhibit good linearity, see Figure 11b. This is in contrast to measurements where
Cld solution was not freshly prepared each time (see our
[engineering page](https://2025.igem.wiki/tudarmstadt/engineering#activity-assay)). When applying linear regression,
the determined KM constant matches the previously reported value. However, kcat is more than twice
as high as expected
[14]. That this is caused by actually higher Cld stock concentration than determined seems unlikely,
as it would contradict both Bradford as well as pyridine hemochrome assay results.
In contrast, the deviation could be caused by varying reaction conditions between this experiment and literature.
Both in our experiments as well as previous reports reaction temperature did not influence initial degradation rates.
Also, differences in phosphate buffer concentrations should not alter activity
[11]. Potentially different expression
and purification methods or spontaneous activity decay, as described earlier, led to lower determined kcat in
[14].

As can be seen in Figure 11a, exponential regression fits the degradation curve better than linear regression.
Also, initial velocity variance within triplicates shrinks and linearity of the Lineweaver-Burk plot increases
(Figure 11b).
As expected, thereby determined kcat is greater. However, obtained kinetic values cannot be verified as - to
our knowledge - Cld from *Azospira oryzae* GR-1 was only analyzed using linear regression so far
[14].
Nevertheless, applying exponential regression seems more accurate to fit obtained degradation curves. Also, similar
substrate dependence of chlorite degradation and enzyme inactivation rates were reported, which implies inactivation
after a fixed number of turnovers. Thus, in reality the degradation curve should be closely represented by an exponential
function
[4].

### Native mass spectrometry

To investigate oligomeric state of Cld it was subjected to Taylor/non-Taylor native mass spectrometry (TNT-MS)
[15]. Using this method,
it was possible to verify that Cld forms homopentamers in solution and our purified Cld has full heme-occupancy. Interestingly, a homodemameric Cld species was also observed

#### Read about native MS analysis of Cld

In native MS, proteins are ionized by electrospray ionization (ESI) preserving non-covalent interactions
such as protein complexes. Subsequently mass-over-charge ratio (m/z) of these ions is determined, in our case
by evaluating their flight time in vacuum. By analyzing two neighboring charge states of one molecular species,
its mass can be derived. (If x1=m/z and x2=m/(z+1), charge state can be calculated by
z=x2/(x1−x2). Subsequently mass can be derived using m=x1·z.)
From that, oligomeric state as well as *e.g.* bound ligands can be identified, as the masses of both
ligand and monomeric *apo*-protein are known
[16].

Here TNT-MS was used to rapidly buffer-exchange Cld into MS-compatible ammonium acetate buffer. For this the
protein sample was injected into ammonium acetate buffer flowing through a capillary. Because of their higher
hydrodynamic radius, part of the protein sample traverses the capillary faster than small molecules and is thus
buffer-exchanged to enable subsequent MS analysis
[15].

To gain more information, precursor ions can be subjected to collision-induced dissociation (CID) before m/z
measurement. For this, the precursor ions are accelerated into neutral gas. Regarding protein complexes this
usually yields asymmetric charge partitioning, meaning dissociation of a highly charged monomer from the complex
[17].
Here collision with neutral gas was used to mildly activate precursor ions, thereby stripping off unspecific
adducts and thus increasing accuracy and resolution.

Cld forms homopentamers in solution
[18]. Therefore, signals corresponding to ions with mass of five Cld subunits
and five heme-groups are expected. In addition, CID should result in ejection of monomers and heme-groups.
Mass spectra are shown in Figure 12, calculated masses are displayed in Table 3.

![Native MS spectrum of Cld](https://static.igem.wiki/teams/5634/wiki/results/cld-ms-2.webp)

**Figure 12:** TNT-MS spectra of Cld. **(a)** Comparison of spectra native and with 80 V collision energy between 614 and 620 m/z shows that heme-group is only present in the latter. Data was normalized to a common intensity in both spectra. **(b,c)** Comparison of Cld signals native **(b)** and with 80 V collision energy **(c)**. Cld pentamers, monomers and decamers are visible

Table 3: Cld TNT-MS calculated masses, standard deviations and mass errors derived from 80 V collision energy spectrum.

| Parameter | *apo*-monomer | *holo*-monomer | Pentamer | Decamer |
| --- | --- | --- | --- | --- |
| Theoretical mass [Da] | 29 757.0 | 30 371.5 | 151 857.7 | 303 715.4 |
| Average observed mass [Da] | 29 755.9 | 30 373.1 | 152 204.8 | 304 848.3 |
| Standard deviation [Da] | 0.9 | 3.3 | 92.0 | 119.0 |
| Mass error relative to theoretical [ppm] | 37 | 52 | 2286 | 3730 |

Without collision energy, charge states belonging to Cld homopentamer with 5 heme groups bound are clearly visible, see Figure 12b. In addition, few *holo*-monomers and surprisingly decamers can be observed .
Employment of 80 V collision energy increases resolution and results in expected ejection of heme-group as well as highly charged *holo*- and predominantly *apo*-monomers, yet pentamer and decamer are still clearly visible. Masses of monomers could be measured with good accuracy, see Table 3. Considerably larger observed masses for oligomers can be explained by unspecific salt/water adducts typical for large ions in native MS
[16].

Overall TNT-MS was able to validate that Cld forms homopentamers in solution. That only fully heme-bound species and no free heme was observed without CID, indicates full heme occupancy, complementary to results from pyridine hemochrome assay.
The observation of a decameric species is interesting, as it is not discussed in the existing literature even though it has already been detected by native MS
[18]. As its peak intensities are very small compared to Cld pentamer, it is less abundant. However, high m/z-ions also get poorly transmitted through the instrument
[16]. Cld decamers seem to be present in solution and could potentially even play a biological role.

We would like to thank M. Sc. Christian Hake and Lermyte group from TU-Darmstadt greatly for providing their TNT-MS instruments, performing MS-experiments together with us as well as annotating and helping us interpret MS data.

#### Conclusion

Chlorite dismutase is of critical interest for (per)chlorate and chlorite bioremediation as it catalyzes the rapid detoxification of chlorite into harmless chloride and oxygen
[4]. In addition, it can be exploited to create oxygen for different applications
[19,  20], which might also prove useful for future space missions
[21].
Here we show first successful Cld production and purification by an iGEM team. We investigated and optimized a spectroscopic activity assay that was performed on a Nanocolor UV/Vis II photometer, designed for completely different applications. It is faster and easier to perform compared to discontinuous methods like iodometric titration
[4]. As many labs already have a photometer capable to measure kinetics, this method is hopefully accessible to many iGEM teams eradicating the need to invest into highly expensive Clark-oxygen sensing electrodes.
Using this assay, it was possible to evaluate Cld kinetics as well as turnover numbers. However, it is not as accurate as alternative methods due to poor time and chlorite concentration resolution.
In addition, it could be verified that AoCld forms homopentamers in solution by native MS. Interestingly, a decameric form also seems to be present. Further research into its abundance and potential biological role seem highly interesting.

#### Impact on project

Research into Cld verified its successful heterologous expression and rapid chlorite degradation
[4], crucial because of chlorite’s cytotoxicity
[22]. These results indicate Cld could work to ensure efficient perchlorate degradation in *B*. *subtilis*. However, irreversible Cld inactivation would probably reduce degradation efficiency. As organisms like *A*. *oryzae* GR-1 are able to grow with perchlorate as sole electron acceptor nevertheless, probably *B*. *subtilis* would be viable as well. Still it could be considered to produce hypochlorite scavengers, which have been shown to increase Cld turnover numbers, like methionine
[10] or guaiacol
[4] *in* *situ* to increase degradation efficiency.

## Chlorite dismutase monomerization

### Design

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
[18].
As suggested by [Prof. Dr. Viktor Stein](human-practices#stein), we investigated the feasibility of obtaining an active monomeric form of this protein.
Our goal was to enable the fusion of monomeric Cld with perchlorate reductase (PcrAB), producing a single multifunctional
enzyme capable of both reducing perchlorate and immediately detoxifying the resulting chlorite.

This coupling minimizes diffusion delays, improving overall efficiency
[22].
While Cld’s enzymatic activity is independent of its oligomeric state, its native pentameric structure poses practical
challenges for fusion. Accommodating pentamerization would require additional design constraints such as linker
optimization and interface stabilization. A monomeric variant simplifies genetic fusion, improves predictability, and
reduces the risk of misfolding or aggregation.

To express our [designed constructs](https://2025.igem.wiki/tudarmstadt/engineering#monomerization), we cloned stratigically mutated sequences into the pQE expression vector, transformed *E. coli* BL21 cells,
and induced expression with IPTG. After lysis, soluble and insoluble fractions were analyzed via SDS-PAGE
[23].

#### Soluble fractions

![SDS-PAGE of Cld monomerization attempts](https://static.igem.wiki/teams/5634/wiki/results/cld-monomerization-sds.webp)

**Figure 13:** SDS-PAGE of soluble cell lysate fractions from *E. coli* BL21(DE3) expressing designed Cld monomer variants (D2-D4, Cld-GFP). Cld wild-type (QCld, mass of 29.8 kDa) was applied as positive control.

#### Insoluble fractions

![Cld monomerization pellet fraction](https://static.igem.wiki/teams/5634/wiki/results/cld-monomerization-sds-pellet-fraction.webp)

**Figure 14:** SDS-PAGE of insoluble cell lysate fractions from the same experiment. Cld monomer designs (D2-D4, Cld-GFP) and Cld wild-type (QCld).

### Interpretation & Discussion

After IPTG induction, only the wild-type Cld (QCld) sample shows a visible Cld band in the soluble fraction.
No detectable expression was observed for designs 2–4 or the Cld-GFP fusion, indicating unsolubility of these variants.

Insoluble Cld bands are visible for the wild-type (QCld) and design 2 samples (see Figure 14), suggesting that design 2 is expressed but insoluble.
Figures 13 and 14 illustrate that, except for design 2, none of the engineered constructs produced detectable amounts of Cld.
In design 2, Cld was visible only in the insoluble fraction, suggesting misfolding or structural disruption.

The mutations may have interfered with the native pentameric assembly of Cld but were not sufficient to induce solubility of
the monomer. For future work, it would be important to revisit the induction protocol, potentially lowering expression
temperature to improve folding. Reducing the number of mutations and focusing on targeted changes could clarify the role of
specific residues. Although the outcome did not meet expectations, it provided valuable insight into the complexity of
protein engineering and the importance of optimizing both design and experimental conditions.

## Cld expression in *B. subtilis*

##### Summary

Our growth curve assay revealed no effect of the pMK4 *cld* construct in *Bacillus subtilis* when exposed to 0-8 mM chlorite, compared to the empty vector control.

### Expectations

To evaluate the functionality of our *cld* expression construct, we aimed to test whether the production of chlorite degrading Cld in *Bacillus subtilis* would indeed improve tolerance against chlorite. Comparing growth under different chlorite concentrations was expected to reveal whether the enzyme can effectively protect the cells.

### Results

Our growth curve assay across 0-8 mM chlorite showed no protective effect of *cld*, as the growth of *Bacillus subtilis* carrying pMK4 *cld* was not significantly higher than that of the empty vector control, as seen in Figures 15 - 23.

# Plotted Growth curves

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"caption":" **Figure 15:** Growth curve of *Bacillus subtilis* at 0 mM chlorite. OD600 was measured for 360 minutes. "
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"caption":" **Figure 16:** Growth curve of *Bacillus subtilis* at 1 mM chlorite. OD600 was measured for 360 minutes. "
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"caption":" **Figure 17:** Growth curve of *Bacillus subtilis* at 2 mM chlorite. OD600 was measured for 360 minutes. "
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"caption":" **Figure 18:** Growth curve of *Bacillus subtilis* at 3 mM chlorite. OD600 was measured for 360 minutes. "
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"caption":" **Figure 19:** Growth curve of *Bacillus subtilis* at 4 mM chlorite. OD600 was measured for 360 minutes. "
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"caption":" **Figure 20:** Growth curve of *Bacillus subtilis* at 5 mM chlorite. OD600 was measured for 360 minutes. "
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"caption":" **Figure 21:** Growth curve of *Bacillus subtilis* at 6 mM chlorite. OD600 was measured for 360 minutes. "
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"caption":" **Figure 22:** Growth curve of *Bacillus subtilis* at 7 mM chlorite. OD600 was measured for 360 minutes. "
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Overall, the growth rate of the pMK4 *cld* strain was never higher than that of the empty vector control, as can be seen in Figure 24. Only for 5 mM chlorite growth rate difference was significant, see Table 4.

![Growth rate comparison of Bacillus subtilis with pMK4 cld and empty vector](https://static.igem.wiki/teams/5634/wiki/engineering/cloning/growth-rates-with-increasing-chlorite-concentrations.webp)

**Figure 24:** Growth rates [h-1] of *Bacillus subtilis* carrying the empty vector pMK4 or the *cld* construct under increasing chlorite concentrations (0–8 mM). The values represent the mean growth rates calculated from three biological replicates.

#### Statistical comparison of growth

**Table 4:** p-values from statistical comparison of growth rates of
*Bacillus subtilis* carrying the empty vector pMK4 and the pMK4 *cld* construct under
increasing chlorite concentrations (0-8 mM). p-values were calculated using a two-tailed Student's t-test.

| Chlorite concentration | 0 mM | 1 mM | 2 mM | 3 mM | 4 mM | 5 mM | 6 mM | 7 mM | 8 mM |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
| p-value | 0,890 | 0,313 | 0,198 | 0,226 | 0,056 | 0,011 | 0,060 | 0,268 | 0,086 |

#### Read discussion

Several factors may explain the absence of a growth advantage of our engineered strain.
One possibility is that chlorite dismutase may not have been functionally active under the tested conditions.
Although Cld is known to be highly effective in chlorite degradation
[5
,14], its expression in our construct may have been insufficient. For example, if the chosen promoter was not active in *Bacillus subtilis* under chosen culturing conditions.
Moreover, as the enzyme is heme-dependent
[18], incomplete heme incorporation or insufficient maturation could, in principle, have led to an inactive protein unable to degrade chlorite. However, this explanation is less likely, since Stefan Hofbauer confirmed that *Bacillus subtilis* is able to synthesize heme *b* and thus assemble active *holo*-chlorite dismutase (see [Human Practice](https://2025.igem.wiki/tudarmstadt/human-practices))
[24].
On the other hand, Cld enzymes usually fulfill their function in the periplasm [3], while our construct was expressed in the cytosol without an *B. subtilis* export sequence.
This mislocalisation may have prevented the enzyme from degrading chlorite in the medium. Strong heterologous expression could also have imposed a metabolic burden on the cells, further reducing growth.
Another important consideration is the quality of the cultures themselves.
During the experiment, we observed unexpected growth patterns in the controls, suggesting that contamination or culture impurity cannot be excluded. For example, the pMK4 *cld* culture may not have been fully homogeneous. Such issues would compromise comparability and may have masked potential protective effects.

### Conclusion

Overall, our data indicate that expression of *cld* in *Bacillus subtilis* under the current construct and/or assay setup did not improve chlorite tolerance. This outcome did not meet our initial expectations, as we anticipated that expression of chlorite dismutase would provide a growth advantage in the presence of chlorite.

The absence of such an effect is likely explained by a combination of biological factors, such as enzyme inactivity, mislocalisation or cellular burden and technical issues, including possible contamination.

#### Future & Applications

These findings highlight the need for improvement in future experiments and applications.
A first step will be to verify *cld* expression.
For example by using a western blot or by fusing the enzyme to GFP, in order to confirm that the promoter is functional and the protein is expressed in *Bacillus subtilis*.
In addition, the cellular localization of Cld should be examined.
This can be done by performing a western blot to determine whether it is solubly expressed and if it reaches the intended compartment.
Since chlorite dismutase functions naturally in the periplasm, directing the protein to the cell surface may allow it to encounter chlorite directly in the medium, thereby enhancing detoxification efficiency.
To further test if heme incorporation is limiting, heme can be supplemented to cultures or lysates and the effect on Cld activity can be measured.
Finally, stricter culture handling and quality control will be important, as contamination or heterogeneity between replicates may have masked potential effects.

## Perchlorate Degradation Assay

#### Goal

We aimed to evaluate the perchlorate degrading capabilities of *Azospira oryzae* GR-1 and *Bacillus subtilis* 168 using a low-cost organic phase extraction method to quantify perchlorate reduction efficiency.

### Results and discussion

Starting from 200 ppm perchlorate, *A. oryzae* wild type (WT) showed no decrease in perchlorate concentration over 7 days of incubation, see Figure 25.
Although it is known as a natural perchlorate reducer [25], our results suggest that critical growth parameters, such as trace element composition, ionic ratios, or redox balance were not met, despite the use of degassed media and mineral oil overlays.

In contrast, *B. subtilis* cultures demonstrated a reduction in perchlorate. After nine days,
the strain carrying the empty pMK4 vector reached ~89 ppm perchlorate (starting from 200 ppm).
Although *B. subtilis* lacks a native perchlorate reductase, its nitrate reductase catalyzes perchlorate
to chlorite conversion [26] (see Figure 25). For the pMK4\_cld vector transformed *B. subtilis* no statement can be made due to high variance between tested replicates.

![Bacillus as our hero](https://static.igem.wiki/teams/5634/wiki/results/resultbacillus-2.avif)

**Figure 25:** Average chloride and perchlorate concentrations measured from *B. subtilis*
(pMK4 and pMK4\_cld) and *A. oryzae* (WT) cultures incubated in M9PT media with 200 ppm acetate and perchlorate at 30°C.
*B. subtilis* data was taken after 9 days cultivation, *A. oryzae* and the media-only control after 7-days (n=2).

To confirm perchlorate conversion, chloride ions were quantified using a [Macherey-Nagel Nanocolor® chloride test kit](https://www.mn-net.com/de/rundkuevettentest-nanocolor-chlorid-200-985019).
With the *B. subtilis* pMK4 strain we could measure an incraese of chloride but given the 1 ppm perchlorate to 0.356 ppm chloride conversion ratio this was not accurately measurable with the kit since the M9PT Media contains more than 1000 ppm chloride ions.

Nevertheless, the trend that *B. subtilis* degraded perchlorate under our conditions might indicate that it is a more robust chassis than *A. oryzae* for perchlorate bioremediation.

### Conclusion

These findings indicate that *B. subtilis*, despite lacking a native perchlorate reductase,
can partially degrade perchlorate through its endogenous nitrate reductase. With the addition of the *cld* gene,
it has the potential to achieve complete perchlorate degradation while avoiding toxic chlorite accumulation. However,
nitrate reductase has a very low perchlorate affinity compared to perchlorate reductase. Thus for complete purification of water
from perchlorate, introduction of perchlorate reductase genes into our chassis would still be necessary [3].

## Outlook

Our results demonstrate significant progress toward a biological system for perchlorate degradation, both as proof-of-concept and as a foundation for future applications.
We achieved the first successful heterologous expression of perchlorate reductase in *E. coli* and verified its activity - an important step toward easier, cheaper enzyme characterication.
Producing this complex enzyme with an affinity tag in a laboratory workhorse like *E. coli* removes the need to rely on slow-growing or hard-to-cultivate native perchlorate-reducing organisms, making the enzyme far more accessible for further research and engineering.
In parallel, we became the first iGEM team to successfully produce and purify chlorite dismutase in *E. coli*, and investigated a simple, low-cost activity assay designed to be accessible to other iGEM teams.
Together, these advances establish a practical platform that lowers barriers for future studies, enabling both academic researchers and future iGEM teams to explore new applications of perchlorate degradation technology.

We also cloned and characterized chlorite dismutase expression in *Bacillus subtilis* 168.
For the final application, genomic integration of the genes of both, chlorite dismutase and perchlorate reductase, can ensure stable expression.

Future work will focus on protein localization using surface display pathways, as these enzymes are naturally found outside the cytoplasmic membrane in perchlorate-reducing bacteria
[27].
Another important direction is enhancing molybdopterin biosynthesis, since it is a critical cofactor in the active site of perchlorate reductase
[3].
These improvements will advance sustainable water purification technology, enabling safe drinking water, hygiene, and agriculture on Mars.

![Astroowl on Mars](https://static.igem.wiki/teams/5634/wiki/results/gazing-into-distance-clay.webp)

Beyond molecular work, we created an affordable, open-source Random Positioning Machine (RPM) that enables the study of biology in microgravity and can simulate Martian gravity on Earth.
Our validated design provides iGEM teams with a powerful new tool to examine the influence of microgravity on parameters like cellular growth or gene expression.
The modular anaerobic bioreactor we built from readily available, low-cost materials enables easy replication and adaptation for different applications.

Looking ahead, we envision our project forming the basis of practical tools for both space and Earth.
For Mars missions, our system could be packaged as an “emergency water kit” a lightweight plastic-bag bioreactor combined with pills containing engineered spores and nutrients that only require water to initiate perchlorate detoxification.
On Earth, our optimized perchlorate detection assay could be adapted into a simple test kit for farmers to monitor soil and water quality in contaminated regions
[28].

Together, these directions bring us closer to building biotechnology that safeguards life not only on Mars, but also here on Earth.

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