Team:TUDelft/Human PracticesWHMN
With our project, Sci-Phi 29, we want to contribute to the field of synthetic biology by allowing easy, cross-species expression of genetic circuits, in a predictable manner.
Such a tool would allow researchers to speed up experiments that would otherwise be very time demanding, and to work with bacterial species that currently can’t be genetically engineered due to a lack of compatible genetic parts. This would not only be helpful to researchers in the field of synthetic biology but it would also reverberate to society. New accessible genetically engineered bacteria could prove useful in industrial processes, bioremediation or in producing new substances such as antibiotics,medicine, bioplastics, biofuels and other biobased products.
In order to carry out this project, we have reached out to researchers, safety experts and the public to refine the design of the Sci-Phi 29 system, for it to truly fit the needs of potentially interested users, whilst making sure the platform is it safe to use.
To do this, we envisioned a hypothetical use case scenario that could help us explore how society could potentially benefit from our project, and what kind of design adaptations would be needed to achieve this. Therefore, we have reached to the general public, industrial companies and (bio)ethicists in search for a real-world problem that could be solved with our system and how such a solution would look like.
Design
Our initial idea
From these interviews we gathered that our design has potential for cross species expression of genetic circuits
We integrated Prof. Daran-Lapujade’s feedback by using different sets of ribozyme insulators, promoters and terminators in order to reduce the chances of homologous recombination phenomena happening within the plasmid
With Jack Pronk’s feedback we decided to develop a modelling tool to find a consensus sequence of codon usage, by gathering extensive data about codon usage in different microbial species
Dr. Chang Liu and Dr. Julian Willis informed us that the expression of these four proteins had to be tightly controlled in prokaryotes. In light of this discovery we redesigned our experiments including a toxicity assay
We came into contact with lots of different people who suggested possible applications for our platform. From these inputs we have created a use case scenario where our system tackles a real world problem.
From multiple conversations it became clear that having proper safety measures for our system is absolutely necessary. Because we want to work as safe and responsible as possible we developed a use case scenario
Input
Dr. Sara Mitri
Dr. Sara Mitri is an Assistant Professor from the Department of Fundamental Microbiology at the University of Lausanne. Dr. Mitri highlighted the usefulness of our system, since her research group works with multiple bacterial species.
She recognized the problem of having to characterize every new organism and thought that the use of the same constructs simplifies a big part of the work in the laboratories like hers, since they are a research groups that work with multiple bacterial species such.
Read MoreDr. Aljoscha Wahl
Dr. Aljoscha Wahl is an Assistant Professor form the Cell Systems Engineering research group, in the TU Delft Department of Biotechnology. He thought that our project could have many potential applications in the field of Biotechnology.
In his opinion our project could have potential for being used in extremophiles for industrial applications. Another possible application would be knocking out genes of non-model bacteria, for research purposes. These specific applications were indicated since these organisms are hard to harness at this moment due to them not being model organisms.
Dr. Wahl’s input helped us understanding there is real potential in allowing cross-species expression of genetic circuits.
Prof. Mark van Loosdrecht
Prof. Mark van Loosdrecht is a Full Professor from the Environmental Biotechnology research group, in the TU Delft Department of Biotechnology. Professor van Loosdrecht remarked the importance of being able to run genetic circuits in poorly characterized bacterial strains, if it were to be done safely.
He remarked that being able to use a wider range of substrates for biotechnological applications could prove very useful. He also said that in Environmental Biotechnology, expressing genetic circuits able to provide an additional selection factor could help in enriching mixed cultures of bacteria.
Prof. van Loosdrecht stated that such a system would prove useful when wanting to isolate specific populations of microorganisms, by providing them with an extra selective advantage, such as antibiotic resistance.
Nonetheless, the use of our plasmid could raise safety concerns, especially if used in microbial communities, due to the existing possibility of horizontal transfer of genes occurring. To gain more knowledge on the matter, we decided to approach safety issues regarding the spread of the plasmid, by getting in touch with safety experts from the RIVM.
Dr. Jean-Marc Daran
Dr. Jean-Marc Daran is an Associate Professor from the Industrial Microbiology research group, in the TU Delft Department of Biotechnology. Dr. Daran remarked the usefulness of having a platform that could allow expressing genetic circuits across different microbial species without the need for remaking them for each organism it has to be expressed in.
He also highlighted that when designing constructs that are meant to work in different microbial contexts, it is important to test these circuits in multiple bacterial species. Nonetheless, Dr. Daran remarked that the first steps to validate the performance of these systems should be in vitro, to make sure that the machinery is indeed working as we expect. Because of this, we decided to prove in vitro replication of the phi29 components next to before trying to express the system in vivo.
Read More
Prof. Pascale Daran-Lapujade
Prof. Pascale Daran-Lapujade is a Full Professor form the Industrial Microbiology research group, in the TU Delft Department of Biotechnology. We learned that homologous recombination could hamper the performance of Sci-Phi 29 if we used the same T7 promoters.
Professor Daran-Lapujade highlighted how this system could work in yeast by compartmentalization of the molecular mechanism to a specific organelle. It could allow protein expression to be more predictable than genome integration, which sometimes results in multiple copies being integrated.
She also noticed how our system could possibly be susceptible to endonucleases and the CRISPR defense mechanism of the host. Because of this, we decided not to include the most common restriction sites in the Sci-Phi 29 sequence in an attempt to reduce the probability of the plasmid being degraded.
On the other hand, we also learned that homologous recombination could hamper the performance of Sci-Phi 29 if we used the same T7 promoters. We integrated Prof. Daran-Lapujade’s feedback by using different sets of ribozyme insulators, promoters and terminators in order to reduce the chances of homologous recombination phenomena happening within our system.
Prof. Jack Pronk
Prof. Jack Pronk is a Full Professor from the Industrial Microbiology research group, in the TU Delft Department of Biotechnology. Jack Pronk informed us that codon usage differs between organisms, and that our system could raise safety concerns.
Professor Pronk suggested that this system could be potentially interesting for developing plug-and-play systems, without the need for integration of the construct into the genome.
Prof. Pronk also mentioned again that a plasmid with orthogonal replication machinery can raise serious safety concerns. This feedback reinforced our intention to contact experts from the RIVM, in order to strengthen the safety aspects of our plasmid.
We also discussed the codon usage in microbial species. Prof. Pronk wisely told us to take it into consideration when expressing exogenous genes in microorganisms. This could make the expression levels vary when expressing Sci-Phi 29 in different microorganisms. With Jack Pronk’s feedback we decided to develop a modelling tool to find a consensus sequence of codon usage, by gathering extensive data about codon usage in different microbial species.
Prof. Richard van Kranenburg
Prof. Richard van Kranenburg is a Special Professor sponsored by Corbion, in Bacterial Cell Factories at the Wageningen University & Research. After learning about the importance of genetic circuits behaving in a predictable way across different bacterial species we applied control systems thinking to design our controllability module. As a result, we structured the Sci-Phi 29 genetic elements as an Incoherent Feed Forward Loop.
We had the opportunity to discuss the multiple steps of characterization of new genetic parts, and the multiple challenges that arise when genetically engineering novel organisms. He explained to us that novel organisms are often limited by the small amount of parts known to them, especially promoters.
Prof. van Kranenburg told us that when expressing genetic circuits in non-model bacteria, those behave in unpredictable ways, even if the parts of the circuit are organism-specific. Each time a genetic circuit is expressed in a different bacterial context there is a need to perfectly define how it behaves in every situation.
Dr. Julian Willis
Dr. Julian Willis is a Postdoctoral Researcher in the David R. Liu research group at the Broad Institute (Cambridge, Massachusetts). Dr. Willis informed us that overexpression of phi29 components can affect the host.
Dr. Julian Willis informed us that the expression of these four proteins had to be tightly controlled in prokaryotes. If the expression is too high, these proteins can interfere with the host’s genome, while if the expression is too low replication might not occur at all. He has been overexpressing proteins from orthogonal replication machinery to identify their toxicity.
Together with Dr. Willis we discussed about exploring different DNA phage replication systems.
Dr. Chang Liu
Dr. Chang Liu is an Assistant Professor from the Department of Biomedical Engineering at the University of California, Irvine. Dr. Liu highlighted the importance of having the expression levels for the phi29 proteins tightly regulated.
Dr. Chan Liu works with the phi29 bacteriophage and its replication machinery. We discussed the challenges of expressing these proteins in vivo, and how to optimize their performance once they are expressed in a microbial context.
Dr. Liu highlighted the importance of having the expression levels for the phi29 proteins tightly regulated, in order to achieve optimal performance of this system, inside the bacterial host of interest. If overexpressed, these proteins will turn out to be harmful to the transformed cell.
An entire experimental module was designed in order to change this, where titration experiments allowed us to find the correct levels of each protein for the system to perform optimally, by using different promoters and concentrations of inducer.
Global Meeting
On August 22nd, we organized a Global Meeting Point together with the International Student Chaplaincy in Delft. The goal of the Global Meeting Point was to bring people from different cultural and religious backgrounds together to discuss a range of cultural, historical, social and environmental subjects.
During the event we explained what synthetic biology is and how we used it to create our project. After our introductory presentation we had an open-ended discussion about GMOs in general but also more specifically about the impact our platform can have on the world, so we could learn people’s opinions on our technology.
From the discussions we learned that the people we talked to are reluctant to release GMOs. Mostly, because they can never anticipate the long term effects these bacteria might have on the ecosystem or people’s health. Another concern was how we would be able to contain our platform if it were to be used in a real world application.
This event helped us to shape our use case scenario . Due to this conversation we implemented physical containment of our platform to a bioreactor in our use case scenario. This physical containment will make sure that our system is not to accidentally influence parts of nature for which it was not designed. Furthermore, we also implemented these design criteria to the safety of our design.
Global Meeting Point gave us the opportunity to learn the opinion of a range of people about the use of synthetic biology. We achieved a better understanding of why people might be reluctant to use genetically modified organisms. While we learned that the public see bacteria as something bad, and dirty, we were very content that we could change some people’s views on bacteria, by showing them they can also be very useful. Click here for a blog post about this event.
Delft Rotary Club
Each of the members from the Rotary Club from Delft is an expert in a different field of society. Researchers, engineers, policymakers, economists, priests and people in the field of humanities compose this diverse club. During discussions with them they raised concerns on the safety of our project.
We discussed our project with the members of our local Rotary Club and we got feedback on the safety aspects of our project. Despite that our project was received very enthusiastically, concerns on the safety were also raised.
When interacting with an audience that is not strictly scientific and talking about synthetic biology, we found that the public is usually reluctant towards safety matters. It became clear that having proper answers and containment measures for our system was not only important for it to be responsible with the world but also for it not to unsettle the general public.
Marjolein Engels
Marjolein Engels is a Senior Proposals Engineer at Allseas, an offshore contractor specialising in pipelay, heavy lift and subsea construction. In this company, Mrs. Engels coordinates a project for microplastic retrieval. Our talk with Marjolein Engels was focused on learning which was the best way to capture these materials, and in which part of the water cycle were they more easily removable.
At first, we learnt that the main source of microplastic pollution comes from domestic and industrial wastewaters. From here, microplastics are carried by water streams until the oceans.
Once in the oceans, they are found in very low concentrations and retrieving them becomes a very inefficient task. For this, Allseas focuses on the retrieval of microplastics in rivers, right before they meet the sea (and so, when they are still at moderate concentrations but there are no more sources of pollution in the river).
They retrieve these pollutants by using the so-called micro-nets. These are nets with very narrow pores that can capture particles with a diameter larger than 1mm. With these, a part of the microplastic particles can be retrieved and disposed of afterwards.
In these nets, not only microplastics but also organic matter gets captured. Separating these two components is one of the major bottlenecks of microplastics retrieval, because of its cost. Usually (and to avoid this kind of problem), the filtered solids are dried and directly incinerated for energy production.
It is also important to add that microplastics change their chemical composition as time goes by. This is due to the plastics being exposed to oxidation phenomena and UV radiation from sunlight. When retrieving microplastic particles, they might not be chemically identical to the original material they were made of.
From this interview, not only did we learn about how microplastics are currently captured and disposed of, we also learnt that this is a problem with more layers of complexity than we thought before:
- The changing chemical composition of these pollutants can lead to unpredictable effects on the ecosystems they are found in, and ultimately in human health.
- Part of the microplastics are not only derived from big plastic objects being degraded, but from products we use in our everyday life (fibers from toothbrushes or synthetic clothes or micro-beads from exfoliants).
- To solve this, we thought that a system of this sort, containing plastic- eating bacteria, would be able to capture the particles larger than 1mm while at the same time also degrading the smallest ones.
- With the Sci-Phi 29 plasmid, we could run genetic circuits inside these microorganisms in order to produce compounds of interest, such as industrial enzymes. This way, a microplastic capturing facility would not only retrieve microplastic waste from water but also turn it into consumer goods.
Prof. Victor de Lorenzo
Prof. Victor de Lorenzo is a Full Professor from the Systems Biology Programme in the CNB (National Centre for Biotechnology), in Madrid, Spain. Prof. de Lorenzo stated that new genetic tools to use in P. putida would be very interesting and useful.
He is currently working in turning the versatile bacterium Pseudomonas putida into a chassis for biotechnological purposes.
From him we learnt that chassis organisms can always be improved to express genetic circuits in a more predictable way. Prof. de Lorenzo stated that new genetic tools to use in P. putida would be very interesting and useful, especially if they focus on achieving predictable expression of genetic constructs.
Knowing that P. putida is able to degrade certain types of plastic and that it is not yet a fully reliable chassis for synthetic biology, we decided to choose P. putida as the organism that will degrade microplastic pollutants in this use case scenario.
Prof. Neelke Doorn
Prof. Neelke Doorn is a Full Professor in Ethics of Water Engineering at the department of Ethics and Technology of Philosophy, in the TU Delft. Together we discussed the ethical implications of the valorisation of municipal wastewater.
Private gains from publicly managed wastewater is a possible conflict that could arise from turning water pollutants into added-value compounds. Professor Doorn stated that the possibility of people complaining about private gains being made from their wastewater is non discardable.
If this system was to be implemented, it should be kept in mind that water authorities are responsible for the management of wastewaters. If this tool was to be implemented, revenues generated with it would go to water authorities. This would retribute to taxpayers.
Reached this point we proceeded to After researching the basic requirements and ethical considerations of the use case scenario, we envisioned how would a system that uses P. putida to degrade microplastics would look like with more technical detail.
With the previously gathered inputs, we know that microplastics should be retrieved as close to their source as possible, to prevent their chemical modification due to oxidation reactions and UV radiation.
Since the main sources of microplastics are of domestic and industrial origin, wastewater treatment plants were chosen as the place where our system would turn these microplastics into added-value compounds.
From Marjolein Engels we learnt that one of the major bottlenecks when retrieving microplastics was the presence of organic matter in the filters. The filtered water stream should have a very low concentration of organic matter, and for this we immediately thought about the Nereda wastewater treatment technology: this system uses a specific type of sludge that settles faster and more efficiently than others.
Using our system in a Nereda wastewater treatment plant would guarantee that the amount of organic matter captured in the micro-nets is lower enough. To see if our system would be compatible with the operating conditions of Nereda, we reached to Paul Roeleveld.
Korienke Smit
Korienke Smit is a Policy Advisor and Coordinator for Safe-by-Design in Education, at the Netherlands National Institute for Public Health and the Environment, who gave input on our safe-by-design.
After Professor Mark van Loosdrecht encouraged us to take a look into the safety aspects of our project, we joined the RIVM program for elaborating a Safe-by-Design approach to our project.
We identified possible users of Sci-Phi 29, possible uses and misuses for this system (unintended or intended) and the consequences of these. Through carefully analysing these aspects, we designed an infographic about how those potential hazards could be minimized or anticipated in different risk cases.
Boerhaave
As a team, we wanted to learn the opinion of the general public about the topic of synthetic biology. These opinions can then help us to shape certain aspects of our project, such as possible applications of our platform. Especially children can give an unbiased insight, allowing us to look at our project in a refreshing way. To get their opinion about synthetic biology, we got in touch with Museum jeugduniversiteit, an organisation that organizes guest lectures for children within the age range of 8-12 years. This was the perfect platform to introduce kids to the topic of microbes and synthetic biology, and get their input regarding these topics.
On September 15th, we went to the Boerhaave Museum in Leiden, where we gave a special lecture to children. In the presentation we addressed the topic of biology in general, cells; the smallest unit of life and what synthetic biology is and its applications are. We co-presented this presentation with Marijn van den Brink (Bachelor student Life Science and Technology), who taught the topic of ‘How to build a synthetic cell’. To make the lecture more interactive, we incorporated an interactive quiz to gain a better insight in the level of knowledge of our young audience about the topics we just presented. This quiz was also used as a tool to start a small debate. With this debate we obtained an understanding of children's opinions about subjects, like how to prevent the escape of engineered organisms, how to work safely with these bacteria, and how bacteria can be modified for intended misuse, such as the development of bioweapons.
From the debate with the children we learned that the safety of genetically modified organisms was a huge concern. They were worried that harmful bacteria might escape. Since we want to try to provide the children a safe future, we addressed these concerns in the safety of our design (Safe by Design).
Use Case Scenario
To identify problems which our system could solve, we took our project outside the lab and talked to potential stakeholders, the local church community and little kids. As a result of these conversations, we identified real world applications which we could solve using our platform. The most occurring issue which we were confronted with was the microplastic pollution.
Safe-by-Design
Sci-Phi 29 facilitates engineering of a repertoire of bacterial species. It provides a set of genetic parts that can be used to assemble a linear plasmid for expression of genes across bacterial species. We want to emphasize that safety and security are important in every possible case. Here we present four possible scenarios for our platform and how we tackle them with the notion of Safe-by-Design. Each section of the infographic below shows a possible scenario, where our platform is used or misused, and this use can be intended or unintended. Each part of the infograhic contains our recommendations on how to handle the corresponding scenario.
Usage Protocol
To limit irresponsible usage of the linear plasmid, we propose a usage protocol that delineates conditions and scenarios in which the plasmid can be put to use. We recommend that users of the Sci-Phi 29 toolkit adhere to our protocol to prevent escape of the linear plasmid.
The usage protocol contains the following points of attention:
- Strict adherence to barrier design criteria
- Usage limited to biosafety level 1 laboratories
- Usage limited in mixed cultures of unknown composition
Barrier design criteria
The barrier design criteria prevent danger possibly caused by the construct due to its universality.
- Physical containment is to be enforced: The use of the plasmid should be limited to closed systems. Bacteria harboring the linear plasmid must only be cultivated in confined and controlled environments. Products of processes using the linear plasmid must be devoid of bacteria and any waste generated must be treated appropriately (in accordance with proper disposal practices) before disposal. To avoid disrupting ecosystems, usage of the plasmid is not allowed in environmental contexts, outside of the lab and without adequate research having been conducted beforehand.
- Dependence on T7 RNA polymerase: To ensure additional safety, we recommend that the individual components of the plasmid should be expressed under a T7 promoter (or a variant thereof). In such a case, initial expression (kickstarting) of the orthogonal replication is needed. Kickstarting would entail the expression of T7 RNA polymerase under the influence of a promoter specific to the bacteria of interest. Such a design choice separates the bacteria specific promoter from the construct and necessitates the use of an external ‘kickstarter’ plasmid (devoid of an origin of replication, for transient expression of T7 RNA polymerase). The kickstarter plasmid can be changed to increase specificity of initiation and therefore increase safety against introduction into unwanted contaminating organisms.
Biosafety level 1 laboratories
Biosafety level 1 laboratories are authorized to work with well-characterized or studied chemicals and organisms that cause no harm to healthy humans (Laboratory biosafety manual). We therefore recommend that usage of our toolkit is limited to biosafety level 1 organisms. Although this could greatly limit applications of our system, we believe our system is still in the early phase of development and despite careful safety designs and considerations, our plasmid could provide an unintended biological advantage to pathogenic or infectious microbes. Furthermore, we discourage the use of our system in human cells.
Limited usage in mixed cultures of unknown composition
The use of our plasmid system should be avoided in microbial communities of unknown composition. Limiting the usage in microbial communities will prevent dangers associated with selection of pathogenic or infectious organisms in culture.