Our gold and integrated practices became two large components this year as we took into consideration what our user groups needed us to provide and how we could ensure that we would be providing a safe tool. Based on our conversations with educators and members of the DIY Biology community, we decided to focus on education as an application of our cell-free system. To ensure our system would be safe and be available for people to use around the world, we explored the regulations surrounding synthetic biology and cell-free systems. We also discovered a potential dual-use of our system and came up with a solution to this using software.

Education Kit

Taking into account the suggestions we received from the interviews we conducted with educators , we have developed a simple experiment that aligns with the Alberta Biology 30 curriculum [1]. Members of the University of Lethbridge iGEM community have previously looked at how the principles of iGEM aligns with the Alberta curriculum [2]. Derek Masterman, a high school teacher, suggested we use physical indicators to ensure students understand that the processes are separate. To validate the occurrence of transcription, the fluorescence capability of the RNA Mango aptamer was employed. Translation is confirmed based upon the chemical reaction between salicylic acid and the protein BMST1 (Paris Bettencourt 2014 BBa_K1403009 ) to produce a wintergreen scent.

During the development of this experiment, we received feedback from students on the Lethbridge High School iGEM Team (pictured below), Keith Aiken (an education student and member of the Lethbridge iGEM team), and Patrick Shackleford (a biology teacher from Winston Churchill High School). The high school students suggested the inclusion of figure captions to avoid student confusion. Keith sent us the current Alberta curriculum guidelines for us to base our learning objectives off of. Our modules were sent to Patrick and he confirmed that they are aligned with what is currently taught as part of the Biology curriculum (pictured below).

In addition to the transcription and translation protocol, we also developed a biosafety module. We designed this module in response to conversations with the Public Health Agency of Canada. They recommended that students need to understand how to interact with microorganisms at an early age to ensure lab safety and proper procedure in the future. Combining this biosafety module with our transcription and translation protocol resulted in the creation of the Next vivo Education kit!

Next vivo Education Kit Schematic:

Check out the full modules below!

Student's Guide	Teacher's Guide	Biosafety Module

It is our hope that we can develop more experiments that utilize a cell-free system. We plan on working under sub-optimal reaction conditions for these experiments. We plan on doing a more in-depth analysis of how these experiments can also align with STEAM (Science, Technology, Engineering, Arts, and Mathematics) initiatives to complement the Lethbridge High School iGEM Team's proposal. We also wish to develop an intermediate level of protocols to better suit the needs of the DIY Biology community members that we spoke with and to create simpler versions of our experimental protocols.

Professional Development

Within education, professional development (PD) days are defined as a variety of specialized training sessions to help administrators and teachers improve their ability to teach [3]. To better integrate synthetic biology concepts within the existing Alberta curriculum, the teachers we spoke with suggested that PD days would be an effective way to ensure that teachers understand the concepts to effectively teach students. Our team paired up with SynBridge, a makerspace located in the University of Lethbridge, to begin the process of hosting these workshops. Our team will work on designing the workshops to be held on PD days, with SynBridge providing the space to host them and acting as a resource for teachers after our iGEM season has ended.

We met with Emily Wilton, the coordinator for SynBridge, to discuss how we would work on the design of the Professional Development Day workshops. Emily told us that they would like to provide the workshops free of charge or at a subsidized rate for the schools, which help to accommodate the limited teaching budgets. We would also work on the design of experiments to teach, while keeping in contact with teachers to ensure they would meet the curriculum needs.

When we discussed our plans with the Winston Churchill Science Department, the best direction to have the PD days instilled was discussed. They suggested forming a collaborative community. In Alberta, a collaborative community is defined as a group of teachers being able to focus on any one school related topic and is open for any teacher to join. Moving forward in this direction, this would also allow these PD days to be open to the whole school district in Lethbridge.

The teachers are planning on visiting SynBridge facilities on April 9th and plan on using this visit to move forward with the development of more synthetic biology initiatives in their curriculum. We plan on this being a long-term goal to be continued on after our season has ended.

Regulation Research

To understand if synthetic biology products and cell-free systems were regulated around the world, we researched relevant legislation. Table 1 is an overview of the regulations that we could find that are implemented in the four legal systems used as was suggested to us by Ian Andrews . Figure 1 is a map of the world to show which countries fall under one of the four legal systems mentioned. We concluded that some legal systems have more established regulations for the use of products derived using biotechnology. Most countries have in place some form of assessments to ensure that any products produced will not have detrimental effects on the environment and human health. As the use of biotechnology grows, we envision that more protocols and legislation will be enacted on the international level to have a more standardized way of regulating products and how they can be used within the different legal systems.

Figure 1 - Colour-coded depiction of countries by type of legal system [4].

Table 1 - Overview of regulations related to the implementation of biotechnology and organismal based devices in society by legal system [5-41].

Legal System	Civil	Common	Mixed	Religious
Laboratory Safety	General guidelines implemented national frameworks being updated	General guidelines implemented in regulations Committees in place to oversee concerns	General guidelines implemented in regulations Committees in place to oversee concerns	National biosafety frameworks in the process of being developed Have signed the Cartagena Protocol
GMO Regulations	In place, appropriate labelling required in most cases to identify GMO status Generally strict regulations in place (EU, JPN)	In place, usage differs depending on country though most countries allow (notable exception: NZ)	Dependent on country: some follow EU regulations and restrict access while others are in the process of developing legislation with minimal regulations	In place, only allow certain crops, need proper labelling to identify GMO status
Environmental Regulations	Environmental impact assessments used to determine effect on biological diversity Environmental release addressed	Environmental impact assessments used to determine effect on biological diversity Environmental release addressed	Environmental impact assessments used to determine effect on biological diversity Some countries have in place environmental release stipulations	Environmental impact assessments used to determine effect on biological diversity Developing more specific legislation for environmental release
Agricultural Regulations	Regulations mostly relate to field trials	Must be approved by regulatory body to not cause a negative impact on the environment	Dependent on country: some have strict regulations on crops that can be planted Others have regulations in place, but allow a greater crop diversity	Regulations relate to type of crops that can be planted
Health Regulations	Must be approved by governing authority to not have negative impacts	Must be approved by governing authority to not have negative impacts	Must be approved by governing authority to not have negative impacts Some countries are still in the process of developing legislation	In development with the growth of biotechnology general regulations state that new materials generated must be approved

We looked further into the regulations of biotechnology products in Canada and the United and tried to find any piece of legislation that mentioned the regulation of cell-free systems. Within Canada and the United States they do not regulate the methodology of how products are made; they focus on the end product. While we could not find any specific mention of "cell-free", Ian was able to clarify within our interview as to why we were unable to find any direct mentions. However, the piece of legislation that we could find that was the most associated with our project is the Assisted Human Reproduction Act in Canada as it prohibits transplanting human-based materials into non-human life forms. From our research, we have been able to determine that cell-free systems are indirectly regulated by existing legislation. Moving forwards with the rapid growth of the biotechnology industry as a whole, it will be pertinent that those involved with the scientific community work with law-makers to develop regulations that are comprehensive enough to capture the growth of the industry to the best of their abilities.

Table 2- Canadian and American Regulation Comparisons for Cell-Free Systems [7,10,11,13-16,19,21,29,33-35,38,41].

Regulation Area	Canada	United States
Health	Assisted Human Reproduction Act (HC): Focus on IVF and new technologies that can aid in reproduction Transplantation into a non-human life form is prohibited	Federal Food, Drug, and Cosmetic Act (FDA) Public Health Service Act (FDA): All biological products must go through licensing process before introduction
Environment	Canadian Environmental Protection Act (EC and HC): Focus on environmental release and new organism activity Environmental assessment required and notification of agencies	National Environment Policy Act (EPA): Requires environmental assessments to determine the impact on the environment
Food/Pharmaceuticals	Food and Drugs Act (CFIA): Focus on labelling and notification of marketing Director must receive notification of intent to sell modified products including an extensive report	Federal Food, Drug, and Cosmetic Act (FDA) Public Health Service Act (FDA) : Must be approved by FDA if food is an additive, but mislabelled food is prohibited New Drug Application submitted for approval to go to marker
Agriculture	Seeds Act (CFIA) Canada Agricultural Products Act (CFIA): Planting, importation, and transportation regulated Must have proof to not cause harm to humans or the environment	Federal Seed Act (APHIS) Plant Protection Act (APHIS) : Planting, importation, and transportation regulated Must have approval before plant is introduced
Pests	Pest Control Products Act (PMRA): Accepted if there is proof that no human health will not be affected or will have long term effects on the environment	Federal Insecticide, Fungicide, and Rodenticide Act (EPA): Registration process detailing proof no harm to the environment
Microorganisms	Canadian Environmental Protection Act (EC and HC): Undergo screening assessment to identify potential harm to humans and the environment	Toxic Substances Control Act (EPA): Must submit notice of use with information on organism
Animals	Health of Animals Act (CFIA): Prohibited to introduce substances without permit	Federal Food, Drug, and Cosmetic Act (FDA): Focusses more on using novel drugs in animal trials Must fill out application detailing effects on the animal and the environment
Pathogens and Toxins	Human Pathogens and Toxins Act (PHAC): Regulates Risk Group 2-4 organisms Prohibited from producing toxins and pathogens within Schedules without a licence	Toxic Substances Control Act (EPA): Does not classify toxic and non-toxic chemicals Chemicals not on list are prohibited from being produced

Table 2 abbreviations

HC: Health Canada
EC: Environment Canada
CFIA: Canadian Food Inspection Agency
PMRA: Pest Management Regulatory Agency
PHAC: Public Health Agency of Canada
FDA: Federal Food and Drug Agency
EPA: Environmental Protection Agency
APHIS: Animal and Plant Health Inspection Service

Biocontainment

In all living organisms each amino acid is encoded by a three nucleotide codon. Changing which codon corresponds to which amino acid has been of scientific interest for many years but has proved difficult as changing one would require changing them throughout the genome, a non-trivial task. As such, the implications of large scale genetic recoding has never been a concern of biosecurity.

Our cell-free system is quite amenable to recoding as the tRNA anticodon can be altered prior to expression changing what amino acid is coded by each codon (see Biocontainment section in Design). As such we hoped to use this feature to make our Next vivo safe for use outside of laboratories. Changing the tRNA anticodons results in a modified codon table that can be used for encoding DNA or RNA message as an input. Using this modified codon table message prevents environmental contamination as all living organisms will be unable to read the modified message, likewise DNA or RNA inputs from external sources cannot be used by our modified codon system.

However, with our system’s ability to alter the normal interactions that occur to produce the standard code, we can generate modified codon tables. The generation of modified codon tables can provide another feature to our system to make it intrinsically safe. By altering our system to only read a modified code, it prevents environmental sequences from contaminating our system modified inputs from being transferred to living organisms and/or modified inputs from being transferred to living organisms [42]. This prevents accidental environmental contamination being introduced by our system. From the research we conducted within environmental regulations, our system becomes more in-line with current regulations and their aim to ensure that synthetic biology produced products do not have a detrimental effect on the environment.

This season we explored the possibilities of how to generate modified sequences. We developed a codon reassignment tool that allows further research into creating safe orthogonal systems. However, this lead us to consider the dual-use implications of this kind of technology for society, particularly DNA synthesis companies. These companies rely on BLAST to detect any harmful sequences sent in DNA synthesis orders. If modified codon tables were used instead of the standard codon table, the companies would not be able to identify harmful sequences. This gap within the current bioinformatic workflow is an issue with no solution being actively pursued by concerned parties. We have come up with a software suite as a solution to this problem and have been in contact with multiple companies for them to test if they could detect a harmful sequence. It was suggested to us in our biosecurity interviews that this may not have been an issue that DNA Synthesis Companies have had to deal with before. We felt it was in the best interest of the synthesis companies to inform them of what we found.

Please continue to our software page for our proposed solution to this issue!

Achievements

We successfully created cell-free TX-TL education modules, aligned with the Alberta curriculum!

We successfully created a biosafety module based on feedback from The Public Health Agency of Canada

We successfully organized a synthetic biology Professional Development day for Alberta teacher!

We successful created a software suite and contacted DNA synthesis companies to mitigate misuse of our system

References

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[4] Central Intelligence Agency. The World Factbook: Legal System, 2016. Retrieved October 9, 2017, from https://www.cia.gov/library/publications/the-world-factbook/fields/2100.html.
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[33] Pest Control Products Act 2002 (Fed) (CAN).
[34] Plant Protection Act 2000 (Fed) (USA).
[35] Public Health Service Act 1944 (Fed) (USA).
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[41] Toxic Substances Control Act 1976 (Fed) (USA).
[42] Jing Ma, N. and F. J. Isaacs, Genomic Recoding Broadly Obstructs the Propagation of Horizontally Transferred Genetic Elements. Cell Systems, 2016. 3: 199-207.

Team:Lethbridge/HP/Gold Integrated