Team:Thessaly/Safety

Overview

Safety was our number one priority throughout our project, thus our team focused on covering all aspects of safety in a Synthetic Biology project, concerning Safe Lab Work, Project Safety and Data Safety. For the Lab Safety, you can find more information in our Safety Form. For our Project’s Safety, we designed multiple layers of kill switches that focus on Gut Containment, Environmental Containment and ensure elimination of Horizontal Gene Transfer (HGT) events. Finally, on Data Safety we focused on data anonymization and encryption both in the software and in the hardware level.

Lab Safety

Project Safety

Personal and Public Safety

Covid-19 Restrictions

All our actions, both inside and outside the Institution, were always in accordance with the government's Covid-19 guidelines.

•  Our Lab Space was at 50% of maximum stuff occupancy.
•  All the Team, including PIs, Instructor, Advisors and Members are vaccinated against the Covid-19.
•  For maximum prevention of spreading the virus, we conducted 2 rapid tests per week to access the lab.
•  The campus had specific facilities for lunch breaks.
•  We wore a face mask at all times.
•  In the case of illness, the whole lab had to quarantine for a week and its contacts for two weeks.

Safe Lab work at BSL1

Figure 1: BSL1 safety guidelines.

Figure 2: Our clean lab bench.

Figure 3: Protection in Gel room.

Figure 4: Laminar Flow avoiding bacterial contamination.

In case of Emergency

In case of fire

The alarm goes off. Everyone must know where the nearest fire extinguisher, emergency exits, telephone, eye shower and first aid kits are. We were induced in the Emergency protocol by the lab manager, during our first week in the lab.

Notify: Other occupants (yell) and Call 199 Fire Station.
Evacuate: The building where the fire is located.
Isolate: Close lab door and IF SAFE TO DO SO, attempt to extinguish.

Figure 5: Fire extinguishers, eye shower and emergency exits in our lab.

In case the power goes out

Stop your experiments and secure chemicals. Turn off all heat sources and shut the fume hood. DO NOT OPEN the freezer door. When the power returns, check the temperatures in your freezers.

In case of coming in contact with strong acid

If there is skin contact, remove clothing and wash with plenty of water, then go to a hospital. If there is eyes/face contact, use eye wash for 15 minutes holding your eyelids open, then go to a hospital. While working with high concentrations of organic acids, dilute with water and flush the area three times if it is spilled out.

In case of an earthquake

During an Earthquake, no matter your location, cover your head and hold onto a static heavy object, in order to avoid being knocked over.
After an Earthquake, Don’t run, Don’t panic and Don’t use elevator. Move outdoors slowly, away from buildings. Have something with you to eat and drink. Make sure you inform the Building Emergency Coordinator, before leaving.

Waste management

We received lab training on how to work safely and minimize potential risks to laboratory personnel and the environment by learning how to discard different types of waste and how to avoid contamination risk, including Bacterial disposal and Sharps disposal.

Figure 6: Autoclaves in our lab

Introduction

Genetically engineered probiotics have a great potential to contribute to diet related Non Communicable Diseases (NCDs). However, the greatest concern of society is safety. Every new technology inherently carries some risk and uncertainty. Our quest is to create a scalable and iterative design, to eliminate risk as much as possible. To do so, we focused on developing biological and mechanical kill switches for every step in our project, resulting in a safe and complete toolkit for Human and Animal Gut dysbiosis. Our design is a template for other iGEM teams to use when building safe Genetically Modified (GM) probiotic systems.

Capsule

The capsule itself provides the physical borders separating the bacteria from the gut. We incorporated the Safe-by-design technique, adding several layers of safety to ensure maximum safety in the event of a malfunction.

Safe design

•  The capsule’s coating (PDMS) is resistant at low pH, not broken down in the digestive system and friendly to it, like common endoscopic capsules (Mimee et al., 2018).
•  SCFAs diffuse from the gut into the capsule, through a semipermeable membrane. Thus, we overcome the obstacle of osmotic pressure differences ensuring the capsules integrity, as Dr. Beltsios, Professor at School of Chemical Engineering NTUA, The Department of Materials Science and Engineering, pointed to us.

Criteria to choose a capsule kill switch

•  The switch should have a quick response to the inducer.
•  The inducer must be cheap and easy to produce.
•  The kill switch must be inclusive for all geographical regions and conditions.
•  The inducer should be non toxic for human, animals and the environment.

Gut containment

Last year, we proposed the L-arabinose inducible kill switch in the event that the capsule's bacterial tank breaks (Amalthea 2020). This year, we tested the functionality of the L-arabinose inducible promoter BBa_K3866000 and incorporated a separated tank besides the bacterial tank, containing L-arabinose. These tanks are connected via a pressure-sensitive switch. If for any reason pressure builds inside the capsule, the switch is activated and the liquids in the tanks get mixed. Learn more at our Hardware Page.

Environmental containment

Our last year’s kill switch, for the environmental containment via the cold-inducible kill switch was rejected from Prof. Dimitrios Karpouzas, in view of the extensive heat waves. He told us that the cold-inducible switch won’t be applicable to regions with higher temperatures. We replaced it with a knockout strain for the dapA so the bacteria can’t survive without the supplementation of the cell wall component dapA. This switch is an extra layer of environmental containment. This way, the bacteria will die from starvation inside the capsule after its rejection, eliminating a possibility of environmental contamination.

The capsule, after its defecation, will be flushed through the toilet. The capsule cannot be collected after usage, so we had to predict and prevent possible environmental contamination from capsule accumulation. So, we contacted Dr.Panagiotis Karas, an expert on toxic waste management. He highlighted that the capsule, if not collected, will eventually break, and both the batteries and the microchip will be released. He proposed that the capsules should be collected from the trash piles after the regular separation of these types of waste. The capsules should be disassembled, and the batteries and the microchip should be distributed to appropriate recycling companies.

In vitro Trials and Pre-Clinical Trials

Last year our feedback from the judges was truly beneficial as they pointed out that the capsule should be smaller in size to pass through the Gastrointestinal (GI) tract of rats for pre-clinical trials. Keeping that in mind, we tried to reduce the size of the capsule, but it was not enough for rats. We concluded that the capsule will pass the in vitro tests simulating the GI tract as Prof. Adele Costabile, Associate Professor in Nutrition at Roehampton University, suggested, and then bigger animals than rats will be used for the pre-clinical Trials, specifically pigs and dogs.

Probiotic

Our engineered probiotic comes in direct contact with the gut, so we needed to be 100% sure of all the safety aspects of the engineered probiotic. Our initial preference on the final GRAS probiotic strain was E. coli Nissle 1917, but after discussing our safety layers with Dr.Dieter Vandenheuvel, Postdoctoral Researcher at the Laboratory of Applied Microbiology and Biotechnology of the University of Antwerp, pointed out that we will have issues implementing the probiotic in food products, because of the E. coli testing. Our E. coli will be present in food but the test cannot differentiate the pathogenic E. coli from ours (FDA, 2021). Our future final probiotic will be a Lactobacili genus as he proposed, in order to make our engineered probiotic suitable to be implemented in a fermented product like yogurt.

Safe design

•  The strain of the engineered probiotic must be on the GRAS list of probiotic strains.
•  Kill-switches should secure bacteria’s death in a specific time frame after the entrance in the body and before its exit in the environment.

Criteria to choose a probiotic kill switch

•  The inducer molecule of the kill-switch must be non-toxic to humans and animals.
•  The inducer cannot be found naturally in the human and animal body.
•  The inducer must be cheap and easy to produce.
•  It must be stable at the stomach’s high pH and not be broken down at earlier points in the digestive tract.

All Possible Kill switches

During the Design of our project, we listed all the probiotic related kill switches in order to choose the best fit for our probiotic.

Anhydrotetracycline (ATC) inducible kill switch from Team Oxford 2018

Pros	Cons
It worked before in an iGEM team (OmiBiome 2018)	ATC can not be consumed by people on a daily basis and there are no alternative molecules (Dos Santos, H. F.1998)
We can control the death of the bacteria	ATC is expensive
It doesn’t harm neighboring bacteria (Specific death of the genetically modified bacteria, destabilizing their LPS)

Switch activated by quorum sensing   (Silpe, J. E. 2019).

Pros	Cons
Controlled timing of killing	It affects neighboring bacteria
Controlled size of gut colony	Not orthogonal

Auxotrophy system with atypical amino acids (Xuan, W., & Schultz, P. G. 2017).

Pros	Cons
Completely orthogonal (atypical amino acid for a vital protein)	Too complicated (the whole translational mechanism needs to change)
Safe and widely tested	No colonization -> no sustainability (bacteria die after short period upon administration)
	For colonization, bacteria need to be fed with a supplement containing the atypical amino acid
	Bacteria might die before even consumed, if the yoghurt is not delivered immediately after production. Also, the yogurt should contain the atypical amino acids which could be more expensive

Auxotrophy to   dapA   (Leventhal et al., 2020).

Pros	Cons
Simple Knockout	Time of bacterial death is not controlled
Already tested from Synlogic	Continuous administration is needed (no sustainability) because of bacteria's death
	Bacteria might die before even consumed, if the yoghurt is not delivered immediately after production

Cryodeath, cold-activated switch   (Stirling et al. 2017).

Pros	Cons
Νot quantitative reliable	Bacteria with this type of kill switch can not survive in a cold yoghurt till consumption
Well characterized	The Switch does not work as intended over 28oC
Widely used

Carbonic Anhydrase knockout   (Merlin C.et al. 2003).

Pros	Cons
Immediate death after defecation	Complicated (Lamda Red recombineering system required)
Worked in previous iGEM team	Anaerobic reactions are difficult to handle in our lab
	Needs constant supply of bicarbonate in lab and anoxic conditions

Cumate switch   (Choi, Y. J.,et al.2010).

Pros	Cons
Cumate is cheap	Not well characterized
Cumate is a plant derivative
The genes are small in size
Non- toxic to human and animals

Tryptophan switch   (Simon, A. J., & Ellington, A. D. 2016).

Pros	Cons
Tryptophan exists in the body	Not innovative
Yogurt has plenty of tryptophan
Bacteria are killed outside the gut

Acyl CoA gene switch   (Klaus, C., et al. 2013).

Pros	Cons
Acyl CoA exists in the body	These constructs would be large in size
	Acyl CoA is similar to SCFAs so this may cause a false positive outcome

GlcNac switch  (Goodman, 2012)

Pros	Cons
GlcNac is produced in gut from mucus	GI disorders cause mucus decrease, which leads to GlcNac production
We can add it to the yoghurt to ensure the survival of our engineered probiotic
It has good impact in the gut
The proposed constructs would be small in size

Administration

The probiotic must not colonize somewhere else except the gut in order to be fully controlled. It is microencapsulated in alginate beads for proper administration and protection from the gastric juice.

Gut containment

Killing the bacteria during treatment is a sustainable proposal compared to companies that produce live biotherapeutics with very frequent administration due to inability of the probiotics to colonize the gut. Our bacteria containing the cumate kill switch will colonize in the gut and after several days of colonization-treatment the patient should take the inducer cumate to clear out the probiotic. To see more about the cumate switch (BBa_K415202).

Environmental containment

The prolonged time of colonization inside the gut may result in leakage through defecation to the environment. In order to avoid that, we chose to use the tryptophan switch, to ensure that when the probiotic leaks out of the gut that is plenty in tryptophan, it won’t survive. Greek traditional yogurt is high in tryptophan so the probiotic will survive in the final product.

Preventing horizontal gene transfer

The final GM bacteria of the capsule and the GM probiotics will have their genetic constructs integrated in their genome to avoid gene transfer from plasmids. (Thomas & Nielsen, 2005).

References

1. Amalthea: A modular platform for monitoring gastrointestinal health. (2020). https://2020.Igem.org/Team;Thessaly/


2. Choi, Y. J., Morel, L., Le François, T., Bourque, D., Bourget, L., Groleau, D., Massie, B., & Míguez, C. B. (2010). Novel, versatile, and tightly regulated expression system for Escherichia coli strains. Applied and environmental microbiology, 76(15), 5058–5066. doi:10.1128/AEM.00413-10


3. Goodman, C. (2012). A GlcNAc switch. Nature Chemical Biology, 8(10), 809-809.doi: 10.1038/nchembio.1077


4. FDA (2021).BAM Chapter 4. Retrieved 18 October 2021, from https://www.fda.gov/food/laboratory-methods-food/bam-chapter-4-enumeration-escherichia-coli-and-coliform-bacteria


5. Klaus, C., Jeon, M. K., Kaemmerer, E., & Gassler, N. (2013). Intestinal acyl-CoA synthetase 5: activation of long chain fatty acids and behind. World journal of gastroenterology, 19(42), 7369–7373.doi: 10.3748/wjg.v19.i42.7369


6. Leventhal D. et al. (2020). Immunotherapy with engineered bacteria by targeting the STING pathway for anti-tumor immunity. Nature Communications, 11(1). doi: 10.1038/s41467-020-16602-0


7. Thomas, C., & Nielsen, K. (2005). Mechanisms of, and Barriers to, Horizontal Gene Transfer between Bacteria. Nature Reviews Microbiology, 3(9), 711-721. doi: 10.1038/nrmicro1234


8. Merlin, C., Masters, M., McAteer, S., & Coulson, A. (2003). Why Is Carbonic Anhydrase Essential to Escherichia coli? Journal of Bacteriology, 185(21), 6415–6424.


9. OmiBiome: Revolutionising Immunosuppressants. (2018) https://2018.igem.org/Team:Oxford


10. Silpe, J. E., & Bassler, B. L. (2019). A Host-Produced Quorum-Sensing Autoinducer Controls a Phage Lysis-Lysogeny Decision. Cell, 176(1-2), 268–280.e13. doi: 10.1016/j.cell.2018.10.059


11. Simon, A. J., & Ellington, A. D. (2016). Recent advances in synthetic biosafety. F1000Research, 5, F1000 Faculty Rev-2118. doi: 10.12688/f1000research.8365.1


12. Stirling, F., Bitzan, L., O'Keefe, S., Redfield, E., Oliver, J., Way, J., & Silver, P. A. (2017). Rational Design of Evolutionarily Stable Microbial Kill Switches. Molecular cell, 68(4), 686–697.e3. https://doi.org/10.1016/j.molcel.2017.10.033


13. Xuan, W., & Schultz, P. G. (2017). A Strategy for Creating Organisms Dependent on Noncanonical Amino Acids. Angewandte Chemie (International ed. in English), 56(31), 9170–9173. doi: 10.1002/anie.201703553

Project Data Safety

Data safety and anonymization were very important for the application and the hardware part of our project. First of all, on the application layer we ensured that e-AMALTHEA strictly follows the GDPR regulations that are in effect in Europe. Furthermore, we connected our application with a hybrid database to combine the safety advantages of a cloud and a conventional database, consulting Stergiou et al., 2017. The users’ data are shared between these two databases and are fully encrypted with standard, hashing-based algorithms the cloud database provides. On the hardware level, we use the Bluetooth communication protocol to pair our hardware with a mobile device. Bluetooth transmission can be safe, in the sense that its small range minimizes the chances of the system being attacked by a “man in the middle” within radius, and also provides encryption through the AES/CCM algorithms (Kitsos et al., 2003).

Dual-use assessment

Concerns may be raised about a project due to its potential to cause major harm inadvertently or to be used in a way to create significant harm, due to either on-purpose unethical practices, or omission in the application of safety protocols. The potential that research would be misapplied, either purposefully or unintentionally, has long been a source of concern in science. It can have ethical and societal consequences, because it includes not just research communities and public health, but also funders, scientific publication, and public communication (WHO 2020). We participated in the 2021 iGEM Risk Assessment Study for other 4 iGEM projects. We followed the same survey assessing our project and it resulted to be reasonably anticipated, which means that the risk is not extremely unlikely.

GDPR for Videos, Photos and Surveys

During our human practices events and visits we used all the proper COVID restriction measures, wearing masks, washing our hands and keeping distances. Before taking a photograph, everyone was asked for permission, while in videos that included children, parent’s permission was received. Also, at the beginning of questionnaires made for gastroenterologists and patients, we asked for participant’s permission. For the surveys we conducted, we also received permission from our GDPR department of University of Thessaly. We required a consent form from every user in order to use their data for research. Finally to ensure data protection, we anonymized the received data.

References

1. Kitsos, P., Sklavos, N., Papadomanolakis, K., & Koufopavlou, O. (2003). Hardware implementation of bluetooth security. IEEE Pervasive Computing, 2(1), 21–29. doi: 10.1109/MPRV.2003.1186722


2. Stergiou, C., & Psannis, K. E. (2017). Efficient and secure BIG data delivery in Cloud Computing. Multimedia Tools and Applications 76, 22803–22822 . https://doi.org/10.1007/s11042-017-4590-4doi:10.1007/s11042-017-4590-4


3. WHO (2020) What is dual-use research of concern? Retrieved 18 October 2021, from https://www.who.int/westernpacific/news/q-a-detail/what-is-dual-use-research-of-concern

igem.thessaly@gmail.com