Team:Sorbonne U Paris/Contribution



How does our work contribute to the projects of future iGEM teams?

Collaborative handbook

During this year, we had the opportunity to realize partnerships with other teams. One of these partnerships consists in the realization of a handbook [Lien vers la page partnership] concerning the good practices of use of a phototrophic frame in synthetic biology. We worked on its writing with other teams that use photosynthetic organisms in their project. Each team was given parts to write to share their experience. We worked with Chlamydomonas reinhardtii, a microalgae and contributed to 3 parts dedicated to these organisms: Assembly standards, Culture & Growth and Transformation. We were able to transmit to future readers protocols, recommendations and solutions to recurrent problems. In order to provide the most complete information, we also interviewed experts in the use of microalgae. This handbook will be available on the wikis of the participating team. Thus, any future participant or potential participant in iGEM wishing to use a phototroph as a chassis will be able to consult this information in order to better understand the experiments that need to be performed.

Ressources education

The materials we have created to communicate [Lien vers la page communication] about our project are all available on our wiki for future iGEM teams. In particular, we have made available many practical work protocols that can be reproduced during public events or interventions in schools. We have also created informative posts on extremophilic microorganisms that future teams can use as inspiration to design their biological system. Finally, we encourage future teams to take up the concept of "Women in Science" to highlight inspiring women whose discoveries have made history but have unfortunately been overlooked.

How to make an organism radio-resistant

Is it possible to make an organism radioresistant? In this section, we will give you some tips to create, in your turn, an organism to take in your shuttle. We will give the main mechanisms related to the acquisition of radioresistance in organisms. However, beware! Not all of them are easily accessible for iGEMers!

Note: as most radioresistant organisms are archaea or prokaryotes, most examples will be from these two kingdoms. It should also be noted that in natural conditions, very few species are exposed to real radiation. The organisms presented here are therefore not so much radioresistant as polyextrêmophilic, the mechanisms of resistance to different stresses being most often common.

1) Behavioral mechanisms

The first category of mechanisms consists of the set of "behavioral strategies'' put in place by organisms to minimize the impact of ionizing radiation. In algae and cyanobacteria, the best known strategy is the use of gas vacuoles to move through the water column in response to light intensity or nutrient availability [1]. Self-shading is also a commonly used strategy in algae: the larger the volume of an alga, the more shade its neighbors receive.

Finally, microorganisms can take refuge in sediments or biofilms to protect themselves from light or radiation [2]. However, how could an iGEMers achieve a genetic construct to direct the behavior of an individual or an entire community? The challenge is undoubtedly a bit large, and we strongly recommend that iGEMers do not pursue this path.

2) Metabolic strategies

a) Synthesis of photoprotective molecules

One of the main avenues for protection against radiation, especially UV, is the expression of photoprotective molecules such as pigments. Among these, we can mention carotenoids, lipidic compounds with polyunsaturated chains that allow to dissipate the light energy received in the cell as well as to collect it for the photosystems[3][4].

Some of them, such as bacterioruberin from Halobacterium salinarum5 or deinoxanthin from Deinococcus radiodurans [6] have the additional ability to perform redox reactions to limit the impact of reactive oxygen species in the cell, and thus act as ROS scavengers. This class of compounds exists in almost all algae and cyanobacteria.

In Chlamydomonas reinhardtii, it is possible to find neoxanthin, loroxanthin (produced in large quantities at low irradiances), violaxanthin (also at low irradiances), anteraxanthin (produced in large quantities at high irradiances), lutein, zeaxanthin (high irradiances) and β-carotene [5].

Increasing pigment concentrations within the cell may seem like an attractive radiation protection solution, but it is actually to be avoided in photosynthetic microorganisms, as pointed out by B. F. Cordero in his article Enhancement of carotenoids biosynthesis in Chlamydomonas reinhardtii by nuclear transformation using a phytoene synthase gene isolated from Chlorella zofingiensis.

The team succeeded in doubling the production of violaxanthin and lutein by overexpressing the CzPSY gene from Chlorella zofingiensis by nuclear transformation in Chlamydomonas reinhardtii. However, this biosynthesis is part of the metabolism of terpenes (lipids containing one or more isoprene units and most often found in linear form) and has as a precursor geranylgeranyl pyrophosphate, which is unfortunately involved in other terpene syntheses: diverting this precursor to boost the production of carotenoids could therefore have an impact on the synthesis of other terpenes. In this case: gibberilins (growth phytohormones), chlorophylls (assimilative pigments of photosynthetic plants) and quinones (electron transporters found in the thylakoid membrane), which directly impacts photosynthesis and growth in a negative way [7].

However, not all pigments share the same biosynthetic pathway: flavonoids, melanin and phycocyanobilin, to name a few, do not have fatty acids as precursors but amino acids. Again, if your body is photosynthetic, beware of melanin! A dark sheath around your cell would prevent photosynthesis. In fact, it is better to forget about pigments if you work with photosynthetic organisms…

An original solution developed by cyanobacteria (but also some other algae and bacteria) consists in Mycosporine-like amino acids (MAAs), small amino acids able to collect light energy, but also to collect reactive oxygen species. Some MAAs therefore provide protection against other types of stress, such as salinity [8].

b) Synthesis of antioxidant molecules

MAAs are not the only chaperone molecules capable of capturing radiation-induced reactive oxygen species. Many ROS scavenger enzymes have similar functions, the best known being catalase and superoxide dismutase.

These two enzymes allow respectively the dismutation of hydrogen peroxide into oxygen and water, and the dismutation of the superoxide anion into oxygen and hydrogen peroxide [9]. Both of these enzymes require cofactors, which are most often transition metals such as manganese or iron.

hese metals also act alone, as chaperone molecules also involved in the reduction of reactive oxygen species, notably in Deinococcus radiodurans and Halobecterium salinarum [10][11][12]. A recent observation by Adrienne Kish's team also highlights the use of bromide ions by Halobacterium salinarum to build a "salt shield" against ionizing radiation [12].

However, these small metabolites have only been studied relatively recently, and the exact chemical mechanisms involved in dismutation processes are not always elucidated. However, small metabolites are an elegant and effective alternative to more "conventional" radioresistance strategies, as shown in the article MDP: A Deinococcus Mn2+ Decapeptide Complex protects Mice from Ionizing Radiation by Gupta et al [11].

Finally, it should be noted that the possibility of implementing a radioresistance strategy through synthetic biology remains constrained by the time, means and knowledge available to iGEMers.

We will mention again the example of "salt shields", which require important evolutionary adaptations, such as proteins resistant to high salinity, and which is in fact impossible to implement during a synthetic biology experiment in the current state of the discipline. Indeed, the proteins of most chassis would tend to denature at such high salt concentrations [13].

3) Repair

One response to ionizing radiation is DNA repair mechanisms. There are many DNA repair mechanisms, including light repair, dark repair, global genome repair and transcription coupled repair.

Original replication mechanisms occurring during DNA repair can also be found in Deinococcus radiodurans, such as break-induced replication (BIR) or single-strand annealing (SSA)[14].

It should be noted, however, that it is now considered that genome repair is not the primary strategy used by organisms to protect themselves from the effects of ionizing radiation, but that it is on the contrary the protection of the proteome that constitutes the sine qua non condition for the survival of the organism[15] .

Deinococcus radiodurans also has several copies of its genome, which ensures that it remains functional even if it is partially damaged. This strategy is also found in other organisms, notably to protect themselves from desiccation[16].

In conclusion, if many strategies are put in place by organisms to protect themselves from radiation and reactive oxygen species in general, few are really applicable to synthetic biology. These mechanisms are often the result of long term evolution, and involve much more than a single gene. However, the study of radioresistant organisms is of great interest, whether for medical or space research, or for practical applications in the nuclear industry, and synthetic biology could prove to be a valuable tool to better understand these mechanisms.

How to access the library of references cited in this article?

  • 1) Go to the following link:
  • 2) Type "The recipe for radioresistance - bibliographic references" in the "Groups" search bar.
  • 3) Enjoy your reading!
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  • [2] Amador-Castro, F., Rodriguez-Martinez, V. & Carrillo-Nieves, D. Robust natural ultraviolet filters from marine ecosystems for the formulation of environmental friendlier bio-sunscreens. Science of The Total Environment 749, 141576 (2020).
  • [3] Bartley, G. E. & Scolnik, P. A. Plant carotenoids: pigments for photoprotection, visual attraction, and human health. Plant Cell 7, 1027–1038 (1995).
  • [4] Demmig-Adams, B. Carotenoids and photoprotection in plants: A role for the xanthophyll zeaxanthin. Biochimica et Biophysica Acta (BBA) - Bioenergetics 1020, 1–24 (1990).
  • [5] Shahmohammadi, H. R. et al. Protective Roles of Bacterioruberin and Intracellular KCl in the Resistance of Halobacterium salinarium against DNA-damaging Agents. JRR 39, 251–262 (1998).
  • [6] Lemee, L., Peuchant, E., Clerc, M., Brunner, M. & Pfander, H. Deinoxanthin: A new carotenoid isolated from Deinococcus radiodurans. Tetrahedron 53, 919–926 (1997).
  • [7] Cordero, B. F., Couso, I., León, R., Rodríguez, H. & Vargas, M. Á. Enhancement of carotenoids biosynthesis in Chlamydomonas reinhardtii by nuclear transformation using a phytoene synthase gene isolated from Chlorella zofingiensis. Appl Microbiol Biotechnol 91, 341–351 (2011).
  • [8] Oren, A. & Gunde-Cimerman, N. Mycosporines and mycosporine-like amino acids: UV protectants or multipurpose secondary metabolites? FEMS Microbiology Letters 269, 1–10 (2007).
  • [9] Shimizu, N., Kobayashi, K. & Hayashi, K. The reaction of superoxide radical with catalase. Mechanism of the inhibition of catalase by superoxide radical. Journal of Biological Chemistry 259, 4414–4418 (1984).
  • [10] Granger, A. C., Gaidamakova, E. K., Matrosova, V. Y., Daly, M. J. & Setlow, P. Effects of Mn and Fe Levels on Bacillus subtilis Spore Resistance and Effects of Mn 2+ , Other Divalent Cations, Orthophosphate, and Dipicolinic Acid on Protein Resistance to Ionizing Radiation. Appl Environ Microbiol 77, 32–40 (2011).
  • [11] Gupta, P. et al. MDP: A Deinococcus Mn2+-Decapeptide Complex Protects Mice from Ionizing Radiation. PLoS ONE 11, e0160575 (2016).
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  • [13] Vauclare, P., Natali, F., Kleman, J. P., Zaccai, G. & Franzetti, B. Surviving salt fluctuations: stress and recovery in Halobacterium salinarum, an extreme halophilic Archaeon. Sci Rep 10, 3298 (2020).
  • [14] Slade, D., Lindner, A. B., Paul, G. & Radman, M. Recombination and Replication in DNA Repair of Heavily Irradiated Deinococcus radiodurans. Cell 136, 1044–1055 (2009).
  • [15] Daly, M. J. et al. Protein Oxidation Implicated as the Primary Determinant of Bacterial Radioresistance. PLoS Biol 5, e92 (2007).
  • [16] Soppa, J. Polyploidy in Archaea and Bacteria: About Desiccation Resistance, Giant Cell Size, Long-Term Survival, Enforcement by a Eukaryotic Host and Additional Aspects. J Mol Microbiol Biotechnol 24, 409–419 (2014).