Team:Evry Paris-Saclay/Poster

Rosewood
by iGEM Evry Paris-Saclay 2020 team, one of (if not) the most colourful, multicultural and diverse teams in the iGEM 2020 competition!

Alexandre Tardivel¹, Angelyne Saint-Julien¹, Cátia Goncalves Pereira¹, Daniel Rodriguez-Pinzon¹, Doriane Blaise¹, Eva Gomes¹, Guillaume Coquard¹, Hassan Hijazi¹, Jellyssa Benjamin¹, Maëva Cherrière², Maxime Pispisa¹, Melissa Nguevo¹, Micky-Love Mocombe¹, Tristan Reif-Trauttmansdorff¹, William Hamlet¹, Paul Soudier³, Sophia Belkhelfa³, Anna Niarakis⁴, Manish Kushwaha⁴ and Ioana Popescu⁵

¹iGEM Student Team Member, ²iGEM Student Team Leader, ³iGEM Team Advisor, ⁴iGEM Team Secondary PI, ⁵iGEM Team Primary PI



Abstract

Illegal wildlife trade is a scourge that affects biodiversity, destroys the fragile equilibrium of natural ecosystems, leads to accelerated extinction of species, and adversely impacts humankind. Elephant’s ivory, rhinoceros’ horn, tiger’s fur are all well-known examples, but the most trafficked wildlife product in the world is rosewood. To the naked eye, rosewood logs are indistinguishable from other non-protected wood species. However, it can be distinguished at the genetic level with high precision. Here, we are developing cheap, portable and easy-to-use biosensors, based on toehold switches. Our biosensor uses engineered molecular machinery of the common gut bacterium to sense nucleic acid signatures specific to the rosewood tree. We demonstrate how to go from the design to the final application, identifying the trafficked rosewood to the family, phylum, or the species level. Deployment of portable and cost-effective rosewood biosensors will enable on-site surveillance and help to protect this rare and valuable species.
Inspiration
Today, the planet faces great risks for its survival. Global warming, the increasing population, and the ecological crisis are forcing humans to question the future of our own species and others that inhabit the planet. Added to this is the illegal and systematic trafficking of wildlife products which constitutes the final nail in the coffin, aggravating the ecological crisis.
Rosewood is the most trafficked product in the world by volume and value. It is more important than elephant ivory, rhino horns and pangolin scales combined [1–4]. It was for this reason that rosewood became the centre of our project.
The trafficked wood is mainly headed to China, which consumes a lot of this wood to make furniture that traditionally represents a certain wealth for the family (a bed made of rosewood can be worth about $1 million). Forests in Africa, South America and Central America have experienced a radical reduction of their rosewood populations to respond to the high-volume demand [5]. On top of the environmental crisis, deforestation is particularly challenging because the countries involved also endure political conflicts, corruption [6], and poor social conditions [2].

References
[1] Guo E. The fight to protect the world’s most trafficked wild commodity. National Geographic (2019).
[2] Ong S, Carver E. The rosewood trade: an illicit trail from forest to furniture. Yale Environment 360 (2019).
[3] Carver E, Ong S. Can forensics help keep endangered rosewood off the black market? Science News (2019).
[4] United Nations Office on Drugs and Crime. The World Wildlife Seizures (World WISE) database (2016).
[5] Schuurman D, Ii PL. The Madagascar rosewood massacre. Madagascar Conservation & Development (2009) 4.
[6] Scanlon JE. Corruption and illegal trade in wildlife: Addressing the nexus between illegal wildlife and forestry trade and corruption. 6th Session of the Conference of the Parties to the UN Convention Against Corruption, Saint Petersburg, Russia (2015).
The Problem & Our solution
To stop the illegal rosewood trade, a big part of the difficulty lies in the strenuous, slow identification process.
In contrast to the noticeable elephant’s ivory or tiger’s fur, once rosewood is logged, it is impossible to distinguish it from other non-protected wood species by naked eye.

Our goal: Develop a biosensor capable of producing a clear and specific detectable signal in the presence of rosewood genetic material.

Although important, the rosewood illegal trade is not well known. We carried out a survey asking if people knew about rosewood, if they owned it, what it meant to them. A major figure emerged: 70% of people did not know about the trade. It's time for a change!

Our goal: Raise awareness about rosewood illegal trafficking.
Idea
The Rosewood biosensor, based on toehold switches [1]:



Properties of Toehold switches that make them excellent candidates for nucleic acids detection:
  • High dynamic range
  • Cell-free paper-based platform
  • Fast detection (60 to 120 minutes)
  • Low limits of detection (30 nM of trigger RNA)
  • High specificity (differences of up to 3 nucleotides)
  • Designed for various specific applications (for example: diagnosis of various pathogenic viruses like Zika [2], Ebola [3], or Norovirus [4]).

References
[1] Green AA, Silver PA, Collins JJ, Yin P. Toehold switches: de-novo-designed regulators of gene expression. Cell (2014) 159: 925–939.
[2] Pardee K, Green AA, Takahashi MK, Braff D, Lambert G, Lee JW, Ferrante T, Ma D, Donghia N, Fan M, Daringer NM, Bosch I, Dudley DM, O’Connor DH, Gehrke L, Collins JJ. Rapid, low-cost detection of Zika virus using programmable biomolecular components. Cell (2016) 165: 1255–1266.
[3] Magro L, Jacquelin B, Escadafal C, Garneret P, Kwasiborski A, Manuguerra J-C, Monti F, Sakuntabhai A, Vanhomwegen J, Lafaye P, Tabeling P. Paper-based RNA detection and multiplexed analysis for Ebola virus diagnostics. Scientific Reports (2017) 7: 1347.
[4] Ma D, Shen L, Wu K, Diehnelt CW, Green AA. Low-cost detection of norovirus using paper-based cell-free systems and synbody-based viral enrichment. Synthetic Biology (Oxford, England) (2018) 3: ysy018.
The unique Rosewood signatures
By analysing and comparing the genomes of different species we may find signatures unique to them.
In order to run comparative genomics against other wood species we looked for genes that were either specific to the rosewood Dalbergia species or genes that showed enough differences to distinguish between them and other non-protected wood species.
No full genomes of Dalbergia species were previously assembled, but previous studies looked at the task of using phylogenetic analysis to identify Dalbergia signatures specially for conservation purposes [1]. Studying them, we found in that it is possible to distinguish Dalbergia protected species endemic to Madagascar from other woods by leveraging the genetic variation in three chloroplast genes:
  • MatK
  • RbcL
  • TrnL-UAA

References
[1] Hassold S, Lowry PP 2nd, Bauert MR, Razafintsalama A, Ramamonjisoa L, Widmer A. DNA barcoding of Malagasy rosewoods: towards a molecular identification of CITES-listed Dalbergia species. PLoS One (2016) 11, e0157881.
Computational design of Toehold Switches:
1st approach - iGEM Webtool
Using the webtool developed by Hong Kong-CUHK 2017 iGEM team [1].

Workflow:
input = target rosewood sequence → chops it into smaller fragments → embeds them each into a fixed switch sequence → the RNAfold algorithm → predict minimum free energy of:
  • trigger
  • switch
  • switch-domains
  • trigger-switch complex

↓
ranking according to an efficacy score

References
[1] To AC-Y, Chu DH-T, Wang AR, Li FC-Y, Chiu AW-O, Gao DY, Choi CHJ, Kong S-K, Chan T-F, Chan K-M, Yip KY. A comprehensive web tool for toehold switch design. Bioinformatics (Oxford, England) (2018) 34: 2862–2864.
Computational design of Toehold Switches:
2nd approach - Toehold Designer
Using the Toehold Designer, our in-house NUPACK based pipeline [1].

Principle: inverse RNA folding
input = target rosewood sequence embedded into random sequence & defined structures

ranking according to several metrics:
  • Predicted translation initiation rates (TIR)
  • Hamming distances between the predicted and the desired structures
  • Minimum free energies of different switch domains

References
[1] Zadeh JN, Steenberg CD, Bois JS, Wolfe BR, Pierce MB, Khan AR, Dirks RM, Pierce NA. NUPACK: Analysis and design of nucleic acid systems. Journal of Computational Chemistry (2011) 32: 170–173.
Build & Test
From computational design to experiments

The top 3 toehold switches for each gene from the first and from the second generation were selected, for a total set of 18 toehold switches.


Whole cell versions of the sensors and triggers were built for screening purposes. Indeed, using plasmids to produce both the sensor and the trigger RNA in the same E. coli cells allowed us to rapidly select the best candidates by evaluating their specific response and fold change.

Each switch was cloned in a low copy plasmid upstream of sfGFP as reported gene and each trigger in a high copy plasmids. For all constructs we used the strong T7 promoter. So, we carried out all characterizations in a BL21(DE3) E. coli strain.

Results
The sfGFP expression was readily visible under blue light, through an amber filter only in the presence of BOTH switch and trigger.
Quantitative data show that the fluorescent signal of switch-trigger co-transformants was much higher compared to the negative control.
This confirms that this toehold switch works as expected
In vivo screening of the 1st generation toehold switches
6 of the 9 first generation toehold switches worked successfully:
(i) efficiently repress the downstream reporter gene expression in the absence of the cognate trigger and
(ii) release the translation inhibition in the presence of the cognate trigger.

Specific response:
  • 6 / 9 toehold switches are specific: fluorescence signal only appears when the trigger has the correct sequence.
  • 3 / 9 switches that did not work: the fluorescence was detected in the absence of the trigger too.


Fold change:
  • 6 / 9 toehold switches have the fluorescence fold changes up to 130, which is on the same order of performance as some of the best switches reported in literature.
In vivo screening of the 2nd generation toehold switches
3 of the 9 second generation toehold switches worked successfully:
(i) efficiently repress the downstream reporter gene expression in the absence of the cognate trigger and
(ii) release the translation inhibition in the presence of the cognate trigger.

Specific response & Fold change:
  • 3 / 9 toehold switches:
    • are specific: the fluorescence signal only appears when the trigger has the correct sequence.
    • have the fluorescence fold changes up to 40, which is on the same order of performance as some of the best switches reported in literature.
  • 5 /9 toehold switches have a low, but not null fluorescence fold changes (bellow 5)
  • 1/ 9 switch that did not work: the fluorescence was detected in the absence of the trigger too.



Implementation
In order to build a cheap and user-friendly biosensor we envision implementing it in cell-free system.
Briefly, as the name suggests, the molecular interactions are shifted to outside the cell whereby only components essential for expression machinery are kept. To fuel the expression reaction, other external components are added such as: dNTPs, cofactors, ions, and others in addition to our DNA construct. Once combined, the system can be freeze-dried in tubes or embedded on a paper. Adding water will allow the biosensing reaction to proceed yielding colorimetric output only, and only if, rosewood RNA is presented.

adapted from [1].

References
[1] Thavarajah W, Silverman AD, Verosloff MS, Kelley-Loughnane N, Jewett MC, Lucks JB. Point-of-use detection of environmental fluoride via a cell-free riboswitch-based biosensor. ACS Synthetic Biology (2020) 9: 10–18.
Proof of concept
6 of the 9 first generation toehold switches were tested in an E. coli based cell-free system.

5 of them worked successfully:
(i) efficiently repress the downstream reporter gene expression in the absence of the cognate trigger and
(ii) release the translation inhibition in the presence of the cognate trigger.


They show decent fold-change, the Rosewood toehold switch DmTrnL-UAA 1.3 being the most promising one to be pursued for on-field implementation.
Rosewood illegal trafficking implications
During our work on human practices, we read the literature and conducted interviews with several personalities (two scientists, a politician and an environmental activist and a lawyer) to enable us to have a much more comprehensive understanding of the phenomenon of rosewood trafficking and its impact on people and the environment.
Raising awareness about the rosewood illegal trafficking
In addition to the competition and the scientific side, iGEM is also a human adventure. The collaborations that we have been able to make have allowed us to improve our project, to prepare materials to raise awareness about the rosewood illegal trafficking (videos, postcards, meetups, …), but above all to meet others, to make science known to everyone, to discover teams from all over the world, from different ethnic groups and to make connections.
Thank you for that!
Achievements
During this this iGEM project, we have:
  • achieved the first ‘scientific’ goal: develop the biosensors capable of producing a clear detectable signal only in the presence of rosewood genetic material.
  • started the ‘social’ goal: raising awareness about Rosewood illegal trafficking.
Perspectives
From here, our project will be implemented in the real world in several phases:
Acknowledgements
All our gratitude to the Systems and Synthetic Biology Lab (iSSB) of the UMR8030 Genomics Metabolics and the Genopole for hosting the team this year again and for having provided technical support and equipments.

We are thankful to Dr. Annah Zhu, Prof. Alex Widner, Haïdar el-Ali and Dr. Julien Prieur who took the time to answer our questions and give us advice.

Thanks to all our sponsors for fundings and material support.