Free Coli was inspired by a desire to address the growing disparity in scientific participation and research between those with and without access to education, resources and research infrastructure.
However, a project that aims to remove barriers to participation in genetic engineering and synthetic biology must also have global safety and security at the front of mind. Our research and consultation into Free Coli's potential benefits and safety and security considerations focused on three areas:
1) Exploring the role Free Coli could play in meeting the UN's Sustainable Development Goals,
2) Investigating the potential for a naturally transformable strain of E. coli to contribute to antibiotic resistance, and
3) Investigating the adequacy of existing gene technology regulations in ensuring community safety in light of the increasing accessibility of synthetic biology tools.
Our human practices research was informed by consultation with experts and engagement with the wider community through our University of Sydney iGEM Genetic Engineering Attitudes and Accessibility Survey, which was developed to 1) assess attitudes about the safety and usage of synthetic biology and genetic engineering, and 2) better understand community perceptions of accessibility barriers in synthetic biology.
Genetic Engineering Attitudes and Accessibility Survey
Our survey also aimed to gauge the public's level of understanding around genetic engineering and genetically modified organisms, to enable us to better understand how an innovative technology like Free Coli would be received, as well as any resulting increase in synthetic biology accessibility.
Some of the questions we asked were demographics based (see below), while others were Likert scales measuring perceptions and beliefs. Others were free text entries, which while posing unique data cleaning and visualisation challenges, were invaluable in informing our approach.
Figure 1. Demographic summaries of survey participants.
Participants' concerns in regard to GMOs and GMO technology were overwhelmingly focused on their relationship to our health, with words such as 'health', 'human', and 'risks' featuring prominently in free text entries. An awareness of driving factors behind GMO opposition or hesitancy is vital in tailoring education, communication and outreach approaches.
Figure 2. Participants' free text entries' visualised in a word cloud.
We conducted a Pearson's Chi-squared test to assess whether a pre-existing understanding of the definition of a GMO had an impact on a participant's attitude in regard to placing trust in scientists to create safe GMO's. We were able to conclude a dependency (p < 0.05), meaning pre-existing knowledge of the definition of a GMO made a participant more likely not to trust scientists, while ignorance of the definition made participants more likely to trust scientists.
We were also interested in whether a person's belief in a potential increase in synthetic biology accessibility had an impact on participants' trust in scientists, but the correlation was statistically insignificant (p > 0.05).
Interestingly, participants' level of education did not have a statistically significant impact on trust in scientists, although biology education seems to have a greater effect than education in general when it comes to participants' trust in scientists.
The survey also presented participants with a passage outlining common concerns associated with GMOs. Both having a biology education and having a higher education in general had a significant impact on whether participants believed the concerns presented in the passage.
The story so far...
The antibiotic age was heralded by the discovery of penicillin by Fleming in 1929. Where previously prognoses for patients afflicted by bacterial infections were fairly grim, there was now a new 'magic bullet' whose shot would revolutionise the treatment of infection, arriving on the world stage just in time to save countless lives in the second world war.
Unforeseen at the time, however, was the idea that the bacteria may want to fight back against the introduction of these new agents. New antibiotic agents were discovered in the decades ensuing from the 40s, but in the 1960s the first evidence of antibiotic resistance emerged in the form of S. aureus which exhibited methicillin resistance (Jevons, Coe, & Parker, 1963). A new generation of antibiotics such as vancomycin was developed to combat this problem, but bacteria eventually developed resistance to these too.
The dangerous situation which modern medicine faces is the emergence of multiple drug resistant 'superbugs' which have acquired resistance to several antibiotic agents, making them very difficult to treat.
But how did we get here?
The road to multiple drug resistant bacteria has been paved over several human generations, but over thousands and thousands of bacterial generations. After their initial discovery, there came an age of excess coupled with an age of increased global mobility. Suddenly, large quantities of antibiotics were being prescribed to humans, and also being introduced to livestock feeds to pre-emptively protect them from bacterial infection. The implications of Darwin's Theory of Natural Selection were looming, with bacteria now facing extraordinary selective pressure. The very small number of bacteria who possessed antibiotic resistance (for example, through random mutation), were able to prosper due to the negative selection of their weaker brethren.
Key also to the proliferation of antibiotic resistance is horizontal gene transfer (HGT, aka lateral gene transfer) - the ability of bacteria to share DNA amongst themselves. Bacteria can exchange mobile DNA elements encoded with the means to survive antibiotic treatments, which led to whole communities exhibiting resistance.
Hotbeds of selective pressure such as hospitals and livestock farms enabled the selection of resistant strains and accelerated the rate of HGT through something known as the SOS response - increased mobile DNA element production in the face of selective stress (Gillings, 2016). Additionally, partially metabolised antibiotics can find their way into waterways and sewage treatment plants, which provide a perfect environment for bacteria to exchange and release mobile DNA elements. Through mobility in waterways and increased global travel, genes of all kinds, including antibiotic resistance genes, have found their way all over the world (Zhu et al., 2017). It has become clear that HGT is the salient issue, so we'll take you through its exact mechanisms.
Mobile DNA Elements and Horizontal Gene Transfer
For DNA to get from host to recipient, it must a) be produced by a host, b) be translocated to the recipient, and c) be incorporated into the recipient genome (in some cases) (Gillings, 2016).
Mobile DNA elements (MDEs) come in many shapes and sizes. Ghaly and Gillings (2018) suggest that it is helpful to think of them as independent ecological units whose aim is to proliferate at cost to the host cell like a parasite. However you think of them, they come in many shapes and sizes.
Figure 1. A schematic showing the various modes of creation, translocation and uptake of MDEs. Reproduced from M. R. Gillings (2016)
Plasmids are pieces of DNA with their own origins of replication, with genes coding for their own replication machinery. Transposons are sections of a chromosome coding for cellular machinery which allows them to pop out of the chromosome and reintegrate at specific target sites. Integrons are sections of DNA that act like libraries, producing an integrase enzyme which acts as a librarian. Integrons can capture and release gene cassettes (genes with a specific 59 bp marker), but can't necessarily move on their own. They can, however, hitch rides on mobile transposons or plasmids.
Once the DNA has been mobilised it must be carried to another cell. Often this is accomplished by conjugation: the host builds a protein pipe to another cell through which mobile DNA elements can be exchanged. Often, plasmids encode for the conjugation machinery, conferring the recipient with the ability to conjugate further (van Hoek et al., 2011). The plasmid may also carry resistance genes, act like a Trojan horse, carrying transposons to the recipient cell ready to integrate into the new genome.
Antibiotic Resistance and Free Coli
From the outset, we were committed to establishing an open dialogue with antibiotic resistance experts to ensure that safety and security was embedded in our project planning, design and execution.
We consulted with Dr Christopher Harmer, a postdoctoral research associate at the University of Sydney who specialises in the small mobile genetic elements and plasmids that are responsible for the spread of antibiotic resistance genes in Gram negative bacteria like E. coli and A. baylyi. Dr Harmer alerted our team to the potential for one of our target genes, ComM, to be implicated in the spread of antibiotic resistance.
There is a suspected homolog in A. baylyi's cousin, A. baumannii, that contains transposon insertion sites implicated in the spread of antibiotic resistance. Our Head of Wet Lab undertook bioinformatic analysis of the two genes (ComM in A. baylyi and A. baumanii) and confirmed they are indeed homologous with 92% similarity.
Our project design involves placing the A. baylyi genes into E. coli, and therefore likely does not involve compatible transposon mechanisms, reducing the potential risk of a naturally transformable E. coli strain contributing to the spread of antibiotic resistance.
Nonetheless, early awareness of this emerging global problem and the role Gram negative bacteria play allowed us to integrate a number of safety features into our design.
Genetically engineered host organisms can pose a risk to the community and the environment if and when they are able to escape the lab environment. It would be very difficult for this bacterium to be converted into a pathogen from its current status as a very safe lab model organism. Our target E. coli lab strain, JM109, contains many intentional mutations that increase their growth efficiency in a lab environment but make them very poor at surviving in the environment or in the human body.
In addition, the foreign A. baylyi genes will be placed under the control of a cumate-inducible promoter. Sequential insertions will target the resistance gene of the previous insertion so that the strain does not build up an excessive number of antibiotic resistances, but we are still able to select the chromosomal insertions at each stage.
Gene Technology Regulation
Accounting for and addressing the impact of off-target effects is a crucial part of synthetic biology research. Likewise, it was important for our research to anticipate and address any off-target effects of designing a new foundational technology for synthetic biology.
Our project's purpose is grounded in the belief that making science more accessible will make the world a better place. However, this is conditional on ensuring that scientific research is conducted in a safe and ethical way.
Given the potential global implementation of our project to serve its purpose of reducing global research inequity, we were also interested in international gene technology regulations. Our early research revealed that there exists no consensus on best practice gene technology regulation (The Third Review of the National Gene Technology Scheme, 2018). Some nations, for example those within the European Union (EU), regulate gene technology based on both “the technique used and the characteristics of the end product” (Custers, 2017).
As we are an Australian team, we began by researching current Australian regulations on gene technology, which is governed by the Gene Technology Act 2000 and Gene Technology Regulations 2001. The Gene Technology Act 2000 regulates genetically modified organisms in Australia to protect the health and safety of the community and the environment. The Act's purpose is to identify and manage the risks posed by or as a result of gene technology. Reviews of regulations are mandated every five years, and have been conducted in 2006, 2011 and 2018.
The Gene Technology Act 2000 regulates genetically modified organisms in Australia to protect the health and safety of the community and the environment. The Act's purpose is to identify and manage the risks posed by or as a result of gene technology. You can access the Act here to view its definitions of 'gene technology', 'genetically modified organism' and 'GMO dealings'.
Reviews of regulations are mandated every five years, and have been conducted in 2006, 2011 and 2018. The most recent review of Australia's National Gene Technology Scheme in 2018 made twenty-seven recommendations, which can be accessed here.
Four recommendations were particularly relevant to our research, design and proposed implementation.
The Review recommends that:
- Extensions and advancements of gene technology, such as synthetic biology, continue to remain within the scope of the Scheme; and
- A watching brief on synthetic biology should be maintained, to ensure the appropriate level of regulation is applied to future applications of synthetic biology.
A 2015 report from the Secretariat of the Convention on Biological Diversity suggested that existing biosafety risk assessment frameworks are likely to be sufficient to assess the risks of current and near-term applications of synthetic biology (Secretariat of the Convention on Biological Diversity, 2015).
The Review recommends that, to ensure the Scheme's current monitoring and enforcement activities remain adequate:
- Regular reviews of these activities are undertaken;
- Regulatory requirements for working with gene technologies are widely communicated and known, and
- The scope and associated risks of 'DIY biology' activity continue to be monitored.
Gene technology has shifted from being solely the remit of universities, research institutions and large companies, to now being accessible to 'community-based citizen scientists' (Citizen Science).
The review highlighted the potential for increases in accessibility to pose increased risks, "the accessibility of genetic modification tools to the general public increases the likelihood of unlicensed experimentation, and with this, safety concerns arise regarding accidental or intentional misuse" (Commonwealth of Australia [Department of Health], pp. 47).
"The Review identified opportunities for further work to be undertaken to quantify the scope of 'DIY biology' activity, ensure that regulatory requirements are widely known, and to further investigate whether current monitoring and enforcement activities are appropriate for all sectors of the Scheme."
This is a potential policy reform area of special interest to the Free Coli team. As part of our collaboration with other iGEM teams and the synthetic biology research community at large, our team also participated in the Australasian Synthetic Biology Challenge and presented our project at the Synthetic Biology Australasia Annual Conference. During the Q&A segment of the conference, a key question from the community was "how could Free Coli make synthetic biology more accessible to DIY biologists?"
The Review recommends that consideration of benefits (e.g. potential economic, environmental and health benefits) should not be introduced as an element of regulatory decision making at this time.
"Regulation of gene technology in Australia focuses on potential risks posed by, or as a result of, gene technology and how these risks may be mitigated. Any potential benefits which may flow from a GMO are not currently considered in regulatory decision making."
"The Review found that for a potential benefit of a GMO to be a consideration in the future, a state and territory position would be required, rather than the Regulator alone considering these factors"
Conko et al. (2016) found that current regulatory burden on GMOs was increasing, despite the lack of identification of novel or incremental risks. The current legislative and regulatory paradigm in regard to gene technology is weighted towards risk aversion. While we believe comprehensive legislation and regulation is vital to ensure gene technology continues to be used to better the world while minimising risks to the community and the environment, there may be reason to rethink this paradigm as the benefits of synthetic biology increase and the accessibility of its applications are enhanced.
The Review recommends that targeted communications be developed to aid public understanding and confidence in the Gene Technology Scheme and identify the most appropriate body/bodies to deliver communications materials.
Sax and Doran (2019) conducted a study evaluating how missing or conflicting information can create ambiguity and influence decisions the community makes about biotechnology. The study identified a "disconnect between consumer perceptions of risk and scientific assessment of risk".
From our review of current Australian and international gene technology legislation, regulation and governance paradigms, our team identified several areas ripe for further community consultation and policy development. As part of our proposed future work, we believe there is a need to improve the proactive education on gene technology regulation for the wider community, as well as consult them on any potential legislative paradigm changes, such as a shift to considering benefits as well as risks as opposed to the status quo, which does not factor potential benefits to the community and/or environment into regulatory decision making.
The review also raised the issue of policy reform to better cater gene technology regulation to emerging 'DIY' synthetic biologists - a new trend that represents both potential and risk. Further work proposed by our team includes the development of a Gene Technology Regulation and Community Safety Educational Toolkit to educate DIY synthetic biologists on gene technology regulation and safety requirements in Australia. This toolkit could also be used as part of collaboration with future iGEM teams to adapt the toolkit to a multitude of legal jurisdictions around the world.
Integrated Human Practices
Human practice has been the heart of Free Coli from inspiration to proposed implementation. Free Coli was inspired by a desire to address the growing disparity in scientific participation and research between those with and without access to education, resources and research infrastructure. We sought to improve a foundational technology to reposition synthetic biology as a tool for reducing, rather than increasing, inequity in research and development.
Our team researched the UN's Sustainable Development Goals, as well as Australia's Commonwealth Scientific and Industrial Research Organisation's Synthetic Biology Future Science Platform during the ideation stage of our project - this research informed the development of our initial research question, 'Can we improve a foundational technology to increase accessibility to synthetic biology?'.
Our decision to focus our efforts on making synthetic biology's most widely used host organism cheaper and more efficient was cemented by the knowledge that it could be used to accelerate the establishment of research and development capabilities in developing nations, and could therefore assist in the advancement of multiple sustainable development goals (see our Sustainable Development Page for more information).
Integrating Stakeholder & Expert Advice
A project that aims to remove barriers to participation in genetic engineering and synthetic biology must also have global safety and security at the front of mind. From the very beginning, the safety of our research and design was informed by consultation with experts and engagement with the wider community.
Our primary safety concern was investigating the potential for a naturally transformable strain of E. coli to contribute to antibiotic resistance. Our team conducted an extensive literature review on the mechanisms underpinning antibiotic resistance as a global health issue, and we consulted Dr Christopher Harmer, a postdoctoral research associate at the University of Sydney who specialises in the small mobile genetic elements and plasmids that are responsible for the spread of antibiotic resistance genes in Gram negative bacteria like E. coli and A. baylyi. Dr Harmer alerted our team to the potential for one of our target genes, ComM, to be implicated in the spread of antibiotic resistance.
Our consultation with Dr Harmer informed our design for Free Coli. We decided to place exogenous A. baylyi genes into E. coli, therefore significantly reducing the likelihood that our E. coli genome and our imported foreign natural transformation genes have compatible transposon mechanisms that could contribute to the spread of antibiotic resistance. An awareness of the role Gram negative bacteria play in antibiotic resistance allowed us to integrate a number of safety features into our design. Our target E. coli lab strain, JM109, contains many intentional mutations that increase their growth efficiency in a lab environment but make them very poor at surviving in the environment or in the human body. In addition, the foreign A. baylyi genes will be placed under the control of a cumate-inducible promoter. Our novel 'Babushka Blocks' recombineering strategy for multiple insertions features sequential insertions that target the resistance gene of the previous insertion so that the strain does not build up an excessive number of antibiotic resistances, while still facilitating efficient selection of chromosomal insertions at each stage.
Due to the impact of Sydney's COVID-19 outbreak and resulting lockdown, our team could not access our laboratory to perform any wet lab experiments and validate our design. In addition to continuing to develop our theoretical design, strengthened by extensive modelling work, we pivoted our project to focus on education and outreach to advance our goal to inspire the next generation of synthetic biology researchers. Our educational resources and presentations for primary school and high school students were informed by our USyd iGEM Genetic Engineering Attitudes and Accessibility Survey. Our survey indicated that having a biology education and having a higher education in general had a significant impact on whether participants believed the concerns about genetically modified organisms.
In our future work proposal, we have also outlined additional pieces of work to make the future implementation and accessibility of Free Coli safer, including the development of a Gene Technology Regulation and Community Safety Requirements educational toolkit to better educate emerging synthetic biologists on how to practice synthetic biology research in a way that is legal and minimises risk to the community and the environment.
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