# Implementation
The Rosewood biosensor is the first step towards sustainable logging of Rosewood and other endangered species because it represents a high-impact pioneering example of using genomic technologies to maintain biodiversity in underdeveloped countries. It will also contribute to sustain the production of items that carry cultural heritage to certain communities, while simultaneously providing jobs and enhancing progress to other communities in need.
## How does the Rosewood biosensor work?
The biosensor, based on toehold switches (Figure 1), works like a scanner that identifies the biological material by checking for specific sequences of the DNA of a specific organism. Indeed, as highlighted in Figure 1, a toehold switch is an RNA–based device containing a ribosome binding site (RBS) and an ATG start codon embedded in a hairpin structure that blocks translation initiation [[1]](#ref1). The hairpin can be unfolded upon binding of a trigger RNA thereby exposing the RBS and permitting translation of the reporter protein. The binding between the trigger and the switch is dictated by RNA-RNA interactions between the complementary base pairs. In the past, such biosensors have been employed to identify relevant biological material and mutations specific to diseases like cancer [[2]](#ref2) and Zika virus [[3]](#ref3) and more recently the SARS-CoV-2 virus [[4,5]](#ref4).
Figure 1. Toehold switches principle (schematic inspired from iGEM Athens 2018).
For successful implementation of our Rosewood detection tool, a device that is easily usable outside of a lab is required. A cell-free system based device seems a suitable choice, for several reasons discussed below.
However, the biggest challenge we envision is the accessibility and quality of rosewood genetic material to be detected.
### What are cell-free systems?
Cell-free system, as the name implies, does not require the presence of living cells but only their core enzyme of gene expression machinery [[6]](#ref6). Two different implementations of these cell-free systems exist: the lysate based one and the PURE system. The lysate based cell-free system consists of a cellular extract, obtained by lysing living cells, supplemented by a buffer solution containing amino acids, salts, cofactors and energetic compounds [[7]](#ref7). The PURE system [[8]](#ref8) is a defined protein synthesising mix obtained by purifying recombinantly produced components and a buffer solution. Both these systems have been applied for detection of RNA using toehold switches [[3,9]](#ref3). For our device we decided to choose a lysate based cell-free implementation since the high cost of PURE systems were found to be incompatible with the low tech-low cost aim of our sensing device.
Its simplicity, speed, and amenability for rational biodesign has make cell-free systems a key platform for synthetic biology applications, including development of biosensors [[10]](#ref10).
### Why are cell-free systems interesting?
Cell-free systems flexibility and controllability allow a wide range of applications such as recombinant proteins synthesis and added-value chemicals production in addition to the realization of real-time biosensor. The very idea of cell-free allays some ethical issues and public concerns as genetic expression can be achieved through designing circuits *in vitro*
where the machinery has been moved outside living cells. Cell-free systems also overcome the daunting complexity of the compartmentalization and allows a level of freedom in accessing the inner workings of the cells [[11]](#ref11). Furthermore, omitting the selective membrane permeability allows for detection of impermeable analytes which in the natural context cannot cross the cell membrane, like RNA for instance, and also for analytes which are toxic to the cells. Other advantages such as sensitivity, stability, robustness and response time are thoroughly researched in this very recent review [[12]](#ref12).
### How are cell-free-based sensors implemented outside the lab?
To exploit its full potential, cell-free systems are freeze-dried on paper discs or in tubes for storage. The addition of water will rehydrate the system and revive its functionality. This provides an inexpensive, low-tech, and quick applicability for the end user. Golden examples of cell-free paper-based sensors are the ones developed for *in vitro* diagnostics including Norovirus [[13]](#ref13), Ebola [[14]](#ref14), Zika virus [[3]](#ref3), to name but a few.
### Reporters systems in cell-free-based biosensors
In order to have a readable output, a biosensor transducer engages different aspects: biofluorescence, bioluminescence, and colorimetry.
To find an inexpensive way to implement on-site the cell-free based biosensor, its readable output must be easily detected with decent dynamic range. In prototyping, fluorescent reporters, such superfolder green fluorescence protein (sfGFP), are used due to their rapid maturation time (<10 min) [[15]](#ref15) where the fluorescence signal is easily measured using a fluorimeter. However, especially in cell-free systems, it is more favorable to reduce energy consumption, which means minimizing the activity of the cell expression machinery. GFP is relatively large, in the first place, which subsequently needs more energy to be synthesized. Moreover, low levels of GFP are barely detected and depend on the sensitivity of the instrumentation used. This makes GFP fluorescence not a reliable quantitative signal unless however calibrated and relative units are translated into an absolute unit (e.g., fluorescein molecules). While GFP does not require any additional substrate to fluoresce, the instruments used to measure GFP are expensive and require significant training to operate.
In this context, for the biosensor to be field-deployable, fluorescent readout needs to be replaced with a robust signal that is easily recognized by the naked eye. Since we evolved under sunlight, the human eye sees only white light of wavelengths within the visual spectrum. A go-to choice instead of fluorescence is to have a color response. In fact, many colorimetric reporters are efficiently utilized to allow direct visualization of sensor output. For example, the most ever used reporter is the LacZ, which produces a blue pigment upon its action on X-gal. LacZ was used in the construct of Zika virus biosensor just aforementioned [[3]](#ref3). Alternatively, in last year’s iGEM (2019), the EPFL team used catechol 2,3-dioxygenase (CDO) as their colorimetric signal inspired by [[16]](#ref16). CDO is an enzyme that cleaves its substrate, catechol, rendering yellow color. Acetylcholinesterase (AChE) is another possibility. This enzyme hydrolyzes the colourless indoxyl acetate to indigo and the inhibition of its activity in the presence of pesticides was used in a paper-based sensor for monitoring of water quality [[17]](#ref17). Lycopene has also been used for sensing purposes by expressing heterologous pathways in *E. coli* and *S. cerevisiae* using either mevalonate or non-mevalonate pathways [[18,19]](#ref18).
Apart from *in vivo* and *in vitro* systems, paper-based biosensors are also employed for detection of specific targets in biological samples. For instance, gold nanoparticles (AuNPs) were used as paper-based colorimetric probes for analyte detection. This quite interesting biosensing method relies on the AuNP aggregation/dispersion states. Once the AuNPs is dispersed due to the presence of a target molecule, an intense red color on the paper is generated within one minute [[20]](#ref20).
Instead of fluorescent or colorimetric reporters, bioluminescent ones (e.g., NanoLuc, LucFF) have also been used in whole cells as well in cell-free systems. Unlike fluorescence, bioluminescence is detected without the need of excitation by a light source as they are based on enzyme-catalyzed biochemical reactions to generate bioluminescence emission. But on other hand, it resembles colorimetric reporters necessitating a substrate unless the whole gene cassette is present [[21]](#ref21).
To sum up, the use of either of the three optical reporters enumerated above ties with its downstream application. Different sensing features are taken into consideration when assessing biosensors such as limit of detection, response time, input and output dynamic ranges, and output visibility [[12,21]](#ref12).
## How to get access to rosewood RNA/DNA ?
### Nucleic acids extraction from wood
There are five methods described to extract DNA from fresh leaves and herbaria of several species of the genus *Dalbergia* and only one of them allows to obtain a good DNA because it is very difficult to extract it due to the fact that the sheets contain a large number of secondary metabolites which interact with the transcription reactions [[22]](#ref22).
However, our biosensor is devised to detect rosewood nucleic acids in wood samples being logged through the borders. DNA isolation from dry wood is more difficult than that of fresh ones due to its highly degraded nature and the presence of high amounts of secondary metabolites [[23]](#ref23). Nevertheless, some purification methods reported successful DNA isolation from wood samples including DNeasy Plant kit (Qiagen)，hexadecyltrimethylammonium bromide (CTAB), sodium dodecyl sulfate (SDS) and Magnetic Beads based methods, the latter being the most efficient one [[24]](#ref24).
### Toehold switches use RNA as trigger, so if one extracts DNA, it should be converted into RNA: how to do it outside a lab (where no PCR machine is available)?
The dearth DNA expected to be extracted from dry wood has to be amplified to allow better workability of our biosensor. Therefore, a pre-amplification step is inevitable. Inarguably, PCR thermocycling is never a choice outside a lab thus the need for a cheaper and low-tech alternative persists. Further, our toehold switch-based biosensor is designed to detect RNA rather than DNA which adds stepwise more complexity. However, the solution lies in using isothermal amplification techniques such as the Loop-Mediated Isothermal Amplification (LAMP), Recombinase Polymerase Amplification (RPA), Nucleic Acid Sequence-Based Amplification (NASBA) [[25]](#ref25) or the Rolling Circle Amplification (RCA) [[26]](#ref26) which is a more proficient on paper method than the other enumerated techniques. Additionally, a CRISPR-based method has recently emerged as an ultra-sensitive nucleic acid detection platform called SHERLOCK (specific high-sensitivity enzymatic reporter unlocking) [[27]](#ref27) and it has been successfully implemented for detection of the malarial parasite with limitations highlighted in there [[28]](#ref28).
Using the suitable isothermal techniques allows the biosensor to be field-applicable, portable, and overcomes the limitation of low-yield DNA extraction.
## Reliability of our detection system
Our biosensor is designed to only distinguish between *Dalbergia* and other timber trees and not between *Dalbergia* species.
This will raise questions concerning the specificity of the developed biosensors. In fact, the unique DNA sequences upon which the switches were built and designed are highly specific to *Dalbergia maritima* [[29]](#ref29). To rule out any potential cross-reactivity and enhance the specificity of the tool, logic gates and signal amplification feedback loop could be used in a further optimization step.
## Who are the proposed end users?
The Rosewood biosensor has been envisioned to be used by custom officials across the trafficking routes of Rosewood. The customs are highly trained in procedures involving the identification of other illegal materials, mainly drugs and explosives. Consequently, we expect to pair up with teams that routinely follow very strict protocols to control the entry and exit of risk substances. Thus, we confidently expect that the personnel available at the Port Police institutions can be trained easily to perform the few steps necessary to use our biosensor.
However, given the very lucrative earnings that can be derived from Rosewood illegal sellings, we understand that several maneuvers can be devised to make even more challenging the sensing at port stations. Thus, in addition, we envision that local police forces will also be able to go to logging places and use our biosensor *in situ*, as a tool to enforce local regulations on the offenders of illegal Rosewood trafficking.
## How do we envision others using this project?
With our proposed end users in mind, the Rosewood biosensor will help save the Rosewood forests by emplying a portable device that is easy-to-use and generates fast outputs. Port authorities will use our device in a cheap 3D-printed physical device that stores the sensor. Several other containers for genetic sensors based on cell-free paper-based devices have already been devised, as for instance for Zika [[3]](#ref3), Ebola [[14]](#ref14) or Noroviruses [[13]](#ref13). We expect to use a similar design that is rapidly and cheaply assembled. Indeed, to produce low-cost biosensor, a paper-based approach is favorable, neglecting the need for cold-chain storage and distribution.
## How would we implement our project in the real world?
Our project will be implemented in the real world in various phases.
**1. Present phase:** The current phase of the project has two main goals technically and socially. The first ‘technical’ goal is to develop the biosensors capable of producing a clear detectable signal only in the presence of rosewood genetic material. This goal was achieved during this iGEM project (see ["Proof of Concept"](https://2020.igem.org/Team:Evry_Paris-Saclay/Proof_Of_Concept) page of this wiki).
The ‘social’ goal consists in raising awareness about Rosewood illegal trafficking. We dream of communities all around the world that share the same preoccupation as us to cut off the traffic of protected species.
**2. Alpha phase:** Our second goal is to continue developing the prototype until it is responsive under favorable conditions. These favorable conditions include high and concentrated quantities of wood material. In parallel, we will start designing our device.
**3. Beta phase:** Once the prototype is functional, we will send various copies of the product to port authorities across the world on the Rosewood trafficking routes and use their feedback to improve it.
**4. Entry to the market:** Once we have invited communities to make part of our project, we will contact institutions across the Rosewood trafficking routes and crowdfund in order to finish up the device and realize it by adding the final “retouches”.
## What are the safety aspects we would need to consider?
At its core, the device presents no risk to its users. Although it implies genetically engineered parts, the diagnostic kit is an *E. coli*-based cell-free transcription/translation system that can be used outside the lab without risk as it does not contain any living organism.
However, the successful implementation of its capabilities means the disruption of trafficking networks that are tremendously lucrative. Also, this easy-to-use tool is built to aid local governments at locations where there might be low control over illegal activities. The safety aspects needed to consider involve the displacement of teams over zones that could potentially be dangerous.
## What are later applications of our project?
Although the biosensor we are developing focuses on Rosewood, we believe it would be feasible to address similar challenges for other endangered species using the same concept. If this happens, it will be a major disruptive on-site advance in the sentinel surveillance of wildlife and in the preservation of ecosystems.
## Democratising Rosewood conservation
Still, after implementing the tool in real life settings there will be a lot of community effort needed for the education and reforestation of damaged zones. Indeed, as detailed on the ["Human Practices"](https://2020.igem.org/Team:Evry_Paris-Saclay/Human_Practices) page of this wiki, the rosewood illegal trade is a complex situation with environmental, social, economical, cultural, legal, … implications. We envision a future where we plant DNA barcoded trees that are specifically grown for logging purposes. *In situ* and *ex situ* conservation measures could be implemented to preserve genetic diversity of Rosewood species and thus counteract their potential uncontrolled loss [[22]](#ref22).
## 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] Hong F, Ma D, Wu K, Mina LA, Luiten RC, Liu Y, Yan H, Green AA. Precise and programmable detection of mutations using ultraspecific riboregulators. Cell (2020) 180: 1018-1032.e16.
[3] 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.
[4] Boettner B. Harvard Wyss Institute’s nasal swab and toehold switch technologies licensed to Agile Biodetection to facilitate SARS-CoV-2 diagnostic efforts. Wyss Institute2020 Oct 5.
[5] Koksaldi IC, Ahan RE, Kose S, Haciosmanoglu N, Kehribar ES, Gungen MA, Ozkul A, Seker UOS. SARS-CoV-2 detection with de novo designed synthetic riboregulators. medRxiv (2020): 2020.07.28.20164004.
[6] Silverman AD, Karim AS, Jewett MC. Cell-free gene expression: an expanded repertoire of applications. Nature Reviews. Genetics (2020) 21: 151–170.
[7] Sun ZZ, Hayes CA, Shin J, Caschera F, Murray RM, Noireaux V. Protocols for implementing an *Escherichia coli* based TX-TL cell-free expression system for synthetic biology. Journal of Visualized Experiments: JoVE (2013): e50762.
[8] Shimizu Y, Inoue A, Tomari Y, Suzuki T, Yokogawa T, Nishikawa K, Ueda T. Cell-free translation reconstituted with purified components. Nature Biotechnology (2001) 19: 751–755.
[9] McNerney MP, Zhang Y, Steppe P, Silverman AD, Jewett MC, Styczynski MP. Point-of-care biomarker quantification enabled by sample-specific calibration. Science Advances (2019) 5: eaax4473.
[10] Laohakunakorn N. Cell-free systems: A proving ground for rational biodesign. Frontiers in Bioengineering and Biotechnology (2020) 8: 788.
[11] Khambhati K, Bhattacharjee G, Gohil N, Braddick D, Kulkarni V, Singh V. Exploring the potential of cell-free protein synthesis for extending the abilities of biological systems. Frontiers in Bioengineering and Biotechnology (2019) 7: 248.
[12] Zhang L, Guo W, Lu Y. Advances in cell-free biosensors: principle, mechanism, and applications. Biotechnology Journal (2020) 15: e2000187.
[13] 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 (2018) 3: ysy018.
[14] 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.
[15] Pédelacq J-D, Cabantous S, Tran T, Terwilliger TC, Waldo GS. Engineering and characterization of a superfolder green fluorescent protein. Nature Biotechnology (2006) 24: 79–88.
[16] Verosloff M, Chappell J, Perry KL, Thompson JR, Lucks JB. PLANT-Dx: A molecular diagnostic for point-of-use detection of plant pathogens. ACS synthetic biology (2019) 8: 902–905.
[17] Sicard C, Glen C, Aubie B, Wallace D, Jahanshahi-Anbuhi S, Pennings K, Daigger GT, Pelton R, Brennan JD, Filipe CDM. Tools for water quality monitoring and mapping using paper-based sensors and cell phones. Water Research (2015) 70: 360–369.
[18] Ostrov N, Jimenez M, Billerbeck S, Brisbois J, Matragrano J, Ager A, Cornish VW. A modular yeast biosensor for low-cost point-of-care pathogen detection. Science Advances (2017) 3: e1603221.
[19] Shaw WM, Yamauchi H, Mead J, Gowers G-OF, Bell DJ, Öling D, Larsson N, Wigglesworth M, Ladds G, Ellis T. Engineering a model cell for rational tuning of GPCR signaling. Cell (2019) 177: 782-796.e27.
[20] Zhao W, Ali MM, Aguirre SD, Brook MA, Li Y. Paper-based bioassays using gold nanoparticle colorimetric probes. Analytical Chemistry (2008) 80: 8431–8437.
[21] Lopreside A, Wan X, Michelini E, Roda A, Wang B. Comprehensive profiling of diverse genetic reporters with application to whole-cell and cell-free biosensors. Analytical Chemistry (2019) 91: 15284–15292.
[22] Ribeiro RA, Lovato MB. Comparative analysis of different DNA extraction protocols in fresh and herbarium specimens of the genus *Dalbergia*. Genetics and molecular research: GMR (2007) 6: 173–187.
[23] Srivastava A, Fatima T, Hanur VS, Rao MS. An effective wood dna extraction protocol for three economic important timber species of India. American Journal of Plant Sciences (2018) 9: 720–726.
[24] Jianguo FU, Jinliang LIU, Xiaojun Y, Yulin AN, Jiayan LUO. DNA extraction and molecular identification of imported *Dalbergia* wood. Journal of Zhejiang A&F University (2013) 30: 627–632.
[25] Gutiérrez J-C, Amaro F, Díaz S, Martín-González A. Environmental biosensors: A microbiological view. In: Thouand G, ed. Handbook of Cell Biosensors. Cham: Springer International Publishing (2019) 1–22.
[26] Liu M, Zhang Q, Li Z, Gu J, Brennan JD, Li Y. Programming a topologically constrained DNA nanostructure into a sensor. Nature Communications (2016) 7: 12074.
[27] Gootenberg JS, Abudayyeh OO, Lee JW, Essletzbichler P, Dy AJ, Joung J, Verdine V, Donghia N, Daringer NM, Freije CA, Myhrvold C, Bhattacharyya RP, Livny J, Regev A, Koonin EV, Hung DT, Sabeti PC, Collins JJ, Zhang F. Nucleic acid detection with CRISPR-Cas13a/C2c2. Science (New York, N.Y.) (2017) 356: 438–442.
[28] Lee RA, Puig HD, Nguyen PQ, Angenent-Mari NM, Donghia NM, McGee JP, Dvorin JD, Klapperich CM, Pollock NR, Collins JJ. Ultrasensitive CRISPR-based diagnostic for field-applicable detection of *Plasmodium* species in symptomatic and asymptomatic malaria. Proceedings of the National Academy of Sciences of the United States of America (2020) 117: 25722–25731.
[29] Hartvig I, Czako M, Kjær ED, Nielsen LR, Theilade I. The use of DNA barcoding in identification and conservation of rosewood (*Dalbergia* spp.). PloS One (2015) 10: e0138231.
Figure 1. Toehold switches principle (schematic inspired from iGEM Athens 2018).
For successful implementation of our Rosewood detection tool, a device that is easily usable outside of a lab is required. A cell-free system based device seems a suitable choice, for several reasons discussed below.
However, the biggest challenge we envision is the accessibility and quality of rosewood genetic material to be detected.
### What are cell-free systems?
Cell-free system, as the name implies, does not require the presence of living cells but only their core enzyme of gene expression machinery [[6]](#ref6). Two different implementations of these cell-free systems exist: the lysate based one and the PURE system. The lysate based cell-free system consists of a cellular extract, obtained by lysing living cells, supplemented by a buffer solution containing amino acids, salts, cofactors and energetic compounds [[7]](#ref7). The PURE system [[8]](#ref8) is a defined protein synthesising mix obtained by purifying recombinantly produced components and a buffer solution. Both these systems have been applied for detection of RNA using toehold switches [[3,9]](#ref3). For our device we decided to choose a lysate based cell-free implementation since the high cost of PURE systems were found to be incompatible with the low tech-low cost aim of our sensing device.
Its simplicity, speed, and amenability for rational biodesign has make cell-free systems a key platform for synthetic biology applications, including development of biosensors [[10]](#ref10).
### Why are cell-free systems interesting?
Cell-free systems flexibility and controllability allow a wide range of applications such as recombinant proteins synthesis and added-value chemicals production in addition to the realization of real-time biosensor. The very idea of cell-free allays some ethical issues and public concerns as genetic expression can be achieved through designing circuits *in vitro*
where the machinery has been moved outside living cells. Cell-free systems also overcome the daunting complexity of the compartmentalization and allows a level of freedom in accessing the inner workings of the cells [[11]](#ref11). Furthermore, omitting the selective membrane permeability allows for detection of impermeable analytes which in the natural context cannot cross the cell membrane, like RNA for instance, and also for analytes which are toxic to the cells. Other advantages such as sensitivity, stability, robustness and response time are thoroughly researched in this very recent review [[12]](#ref12).
### How are cell-free-based sensors implemented outside the lab?
To exploit its full potential, cell-free systems are freeze-dried on paper discs or in tubes for storage. The addition of water will rehydrate the system and revive its functionality. This provides an inexpensive, low-tech, and quick applicability for the end user. Golden examples of cell-free paper-based sensors are the ones developed for *in vitro* diagnostics including Norovirus [[13]](#ref13), Ebola [[14]](#ref14), Zika virus [[3]](#ref3), to name but a few.
### Reporters systems in cell-free-based biosensors
In order to have a readable output, a biosensor transducer engages different aspects: biofluorescence, bioluminescence, and colorimetry.
To find an inexpensive way to implement on-site the cell-free based biosensor, its readable output must be easily detected with decent dynamic range. In prototyping, fluorescent reporters, such superfolder green fluorescence protein (sfGFP), are used due to their rapid maturation time (<10 min) [[15]](#ref15) where the fluorescence signal is easily measured using a fluorimeter. However, especially in cell-free systems, it is more favorable to reduce energy consumption, which means minimizing the activity of the cell expression machinery. GFP is relatively large, in the first place, which subsequently needs more energy to be synthesized. Moreover, low levels of GFP are barely detected and depend on the sensitivity of the instrumentation used. This makes GFP fluorescence not a reliable quantitative signal unless however calibrated and relative units are translated into an absolute unit (e.g., fluorescein molecules). While GFP does not require any additional substrate to fluoresce, the instruments used to measure GFP are expensive and require significant training to operate.
In this context, for the biosensor to be field-deployable, fluorescent readout needs to be replaced with a robust signal that is easily recognized by the naked eye. Since we evolved under sunlight, the human eye sees only white light of wavelengths within the visual spectrum. A go-to choice instead of fluorescence is to have a color response. In fact, many colorimetric reporters are efficiently utilized to allow direct visualization of sensor output. For example, the most ever used reporter is the LacZ, which produces a blue pigment upon its action on X-gal. LacZ was used in the construct of Zika virus biosensor just aforementioned [[3]](#ref3). Alternatively, in last year’s iGEM (2019), the EPFL team used catechol 2,3-dioxygenase (CDO) as their colorimetric signal inspired by [[16]](#ref16). CDO is an enzyme that cleaves its substrate, catechol, rendering yellow color. Acetylcholinesterase (AChE) is another possibility. This enzyme hydrolyzes the colourless indoxyl acetate to indigo and the inhibition of its activity in the presence of pesticides was used in a paper-based sensor for monitoring of water quality [[17]](#ref17). Lycopene has also been used for sensing purposes by expressing heterologous pathways in *E. coli* and *S. cerevisiae* using either mevalonate or non-mevalonate pathways [[18,19]](#ref18).
Apart from *in vivo* and *in vitro* systems, paper-based biosensors are also employed for detection of specific targets in biological samples. For instance, gold nanoparticles (AuNPs) were used as paper-based colorimetric probes for analyte detection. This quite interesting biosensing method relies on the AuNP aggregation/dispersion states. Once the AuNPs is dispersed due to the presence of a target molecule, an intense red color on the paper is generated within one minute [[20]](#ref20).
Instead of fluorescent or colorimetric reporters, bioluminescent ones (e.g., NanoLuc, LucFF) have also been used in whole cells as well in cell-free systems. Unlike fluorescence, bioluminescence is detected without the need of excitation by a light source as they are based on enzyme-catalyzed biochemical reactions to generate bioluminescence emission. But on other hand, it resembles colorimetric reporters necessitating a substrate unless the whole gene cassette is present [[21]](#ref21).
To sum up, the use of either of the three optical reporters enumerated above ties with its downstream application. Different sensing features are taken into consideration when assessing biosensors such as limit of detection, response time, input and output dynamic ranges, and output visibility [[12,21]](#ref12).
## How to get access to rosewood RNA/DNA ?
### Nucleic acids extraction from wood
There are five methods described to extract DNA from fresh leaves and herbaria of several species of the genus *Dalbergia* and only one of them allows to obtain a good DNA because it is very difficult to extract it due to the fact that the sheets contain a large number of secondary metabolites which interact with the transcription reactions [[22]](#ref22).
However, our biosensor is devised to detect rosewood nucleic acids in wood samples being logged through the borders. DNA isolation from dry wood is more difficult than that of fresh ones due to its highly degraded nature and the presence of high amounts of secondary metabolites [[23]](#ref23). Nevertheless, some purification methods reported successful DNA isolation from wood samples including DNeasy Plant kit (Qiagen)，hexadecyltrimethylammonium bromide (CTAB), sodium dodecyl sulfate (SDS) and Magnetic Beads based methods, the latter being the most efficient one [[24]](#ref24).
### Toehold switches use RNA as trigger, so if one extracts DNA, it should be converted into RNA: how to do it outside a lab (where no PCR machine is available)?
The dearth DNA expected to be extracted from dry wood has to be amplified to allow better workability of our biosensor. Therefore, a pre-amplification step is inevitable. Inarguably, PCR thermocycling is never a choice outside a lab thus the need for a cheaper and low-tech alternative persists. Further, our toehold switch-based biosensor is designed to detect RNA rather than DNA which adds stepwise more complexity. However, the solution lies in using isothermal amplification techniques such as the Loop-Mediated Isothermal Amplification (LAMP), Recombinase Polymerase Amplification (RPA), Nucleic Acid Sequence-Based Amplification (NASBA) [[25]](#ref25) or the Rolling Circle Amplification (RCA) [[26]](#ref26) which is a more proficient on paper method than the other enumerated techniques. Additionally, a CRISPR-based method has recently emerged as an ultra-sensitive nucleic acid detection platform called SHERLOCK (specific high-sensitivity enzymatic reporter unlocking) [[27]](#ref27) and it has been successfully implemented for detection of the malarial parasite with limitations highlighted in there [[28]](#ref28).
Using the suitable isothermal techniques allows the biosensor to be field-applicable, portable, and overcomes the limitation of low-yield DNA extraction.
## Reliability of our detection system
Our biosensor is designed to only distinguish between *Dalbergia* and other timber trees and not between *Dalbergia* species.
This will raise questions concerning the specificity of the developed biosensors. In fact, the unique DNA sequences upon which the switches were built and designed are highly specific to *Dalbergia maritima* [[29]](#ref29). To rule out any potential cross-reactivity and enhance the specificity of the tool, logic gates and signal amplification feedback loop could be used in a further optimization step.
## Who are the proposed end users?
The Rosewood biosensor has been envisioned to be used by custom officials across the trafficking routes of Rosewood. The customs are highly trained in procedures involving the identification of other illegal materials, mainly drugs and explosives. Consequently, we expect to pair up with teams that routinely follow very strict protocols to control the entry and exit of risk substances. Thus, we confidently expect that the personnel available at the Port Police institutions can be trained easily to perform the few steps necessary to use our biosensor.
However, given the very lucrative earnings that can be derived from Rosewood illegal sellings, we understand that several maneuvers can be devised to make even more challenging the sensing at port stations. Thus, in addition, we envision that local police forces will also be able to go to logging places and use our biosensor *in situ*, as a tool to enforce local regulations on the offenders of illegal Rosewood trafficking.
## How do we envision others using this project?
With our proposed end users in mind, the Rosewood biosensor will help save the Rosewood forests by emplying a portable device that is easy-to-use and generates fast outputs. Port authorities will use our device in a cheap 3D-printed physical device that stores the sensor. Several other containers for genetic sensors based on cell-free paper-based devices have already been devised, as for instance for Zika [[3]](#ref3), Ebola [[14]](#ref14) or Noroviruses [[13]](#ref13). We expect to use a similar design that is rapidly and cheaply assembled. Indeed, to produce low-cost biosensor, a paper-based approach is favorable, neglecting the need for cold-chain storage and distribution.
## How would we implement our project in the real world?
Our project will be implemented in the real world in various phases.
**1. Present phase:** The current phase of the project has two main goals technically and socially. The first ‘technical’ goal is to develop the biosensors capable of producing a clear detectable signal only in the presence of rosewood genetic material. This goal was achieved during this iGEM project (see ["Proof of Concept"](https://2020.igem.org/Team:Evry_Paris-Saclay/Proof_Of_Concept) page of this wiki).
The ‘social’ goal consists in raising awareness about Rosewood illegal trafficking. We dream of communities all around the world that share the same preoccupation as us to cut off the traffic of protected species.
**2. Alpha phase:** Our second goal is to continue developing the prototype until it is responsive under favorable conditions. These favorable conditions include high and concentrated quantities of wood material. In parallel, we will start designing our device.
**3. Beta phase:** Once the prototype is functional, we will send various copies of the product to port authorities across the world on the Rosewood trafficking routes and use their feedback to improve it.
**4. Entry to the market:** Once we have invited communities to make part of our project, we will contact institutions across the Rosewood trafficking routes and crowdfund in order to finish up the device and realize it by adding the final “retouches”.
## What are the safety aspects we would need to consider?
At its core, the device presents no risk to its users. Although it implies genetically engineered parts, the diagnostic kit is an *E. coli*-based cell-free transcription/translation system that can be used outside the lab without risk as it does not contain any living organism.
However, the successful implementation of its capabilities means the disruption of trafficking networks that are tremendously lucrative. Also, this easy-to-use tool is built to aid local governments at locations where there might be low control over illegal activities. The safety aspects needed to consider involve the displacement of teams over zones that could potentially be dangerous.
## What are later applications of our project?
Although the biosensor we are developing focuses on Rosewood, we believe it would be feasible to address similar challenges for other endangered species using the same concept. If this happens, it will be a major disruptive on-site advance in the sentinel surveillance of wildlife and in the preservation of ecosystems.
## Democratising Rosewood conservation
Still, after implementing the tool in real life settings there will be a lot of community effort needed for the education and reforestation of damaged zones. Indeed, as detailed on the ["Human Practices"](https://2020.igem.org/Team:Evry_Paris-Saclay/Human_Practices) page of this wiki, the rosewood illegal trade is a complex situation with environmental, social, economical, cultural, legal, … implications. We envision a future where we plant DNA barcoded trees that are specifically grown for logging purposes. *In situ* and *ex situ* conservation measures could be implemented to preserve genetic diversity of Rosewood species and thus counteract their potential uncontrolled loss [[22]](#ref22).
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