Safety Form

2020 UCL iGEM

1. Please upload a photo or two of your lab to the iGEM 2020 server （ include your team name in the file name ） , preferably showing the relevant safety features and paste the link here: (Instructions on how to upload an image to our servers can be found here)

Due to the COVID-19 outbreak, we have not had access to the lab throughout the summer. Therefore, no photos can be provided.

2. Describe the goal of your project: what is your engineered organism supposed to do? Please include specific technical details and names of important parts.

Our project combines plastic degradation with low-cost water desalination in a microbial fuel cell (MFC) coculturing engineered Escherichia coli BL21 DE3, engineered Pseudomonas Putida KT2440, and wild type Shewanella oneidensis MR-1. The main goals are to degrade polyethylene terephthalate (PET)-derived plastic, desalinate saline water, and generate electricity. E. coli and P. putida are engineered to convert PET into lactate uptaken by S. oneidensis to generate bioelectricity capable of desalinating water by transferring electrons from the anode chamber to the cathode chamber.

E. coli will express exogenous PET degrading enzymes, PETase and MHETase, from Ideonella sakaiensis. We have designed a PETase-MHETase fusion protein in Benchling, which has not been documented by any previous iGEM teams in the Parts Registry. E. coli will degrade PET extracellularly by secreting PETase-MHETase fusion protein. As the sole carbon source for E. coli, engineered to not uptake other carbon sources, PET will be degraded into mono (2-hydroxyethly) terephthalate (MHET) by PETase. Next, MHETase will convert MHET into terephthalic acid (TPA) and ethylene glycol (EG). TPA will be the only carbon source for P. putida. EG will be converted into glycolaldehyde which will be uptaken by E. coli to support its biomass growth. From Flux Balance Analysis (FBA), we learned recently that the conversion of glycolaldehyde into pyruvate for E. coli will need to be engineered – the next step for our team. We will update the safety form once more details on which genes we are cloning become available. FBA also provided us with a list of genes to knock out for maximising E. coli's production of EG and TPA, but we are not performing any wet lab experiments to simulate these knockouts this year. Therefore, this year, to facilitate PET degradation and TPA and EG production, we decided to overexpress our PETase-MHETase fusion protein, of which the secretion efficiency to the extracellular environment will be tested with secretion tag lamB. The 2020 Exeter team, with lab access, will help us to perform this test in wet lab.

P. putida will be engineered to uptake TPA as the only carbon source from extracellular environment and act as a mediator by converting TPA into lactate, providing a viable carbon source for S. oneidensis. Three genes from Comamonas sp. strain E6 have been identified to encode TPA transporter: tpiA and

tpiB for encoding TpiA-TpiB membrane components and tphCII for encoding TPA binding protein. Since there is not any well-characterized Biobricks with these genes, our team is designing Benchling constructs that can be used in future years to express. The conversion of TPA to lactate can be achieved in a 2-step process. First, P. putida will convert TPA to protocatechuic acid (PCA) by heterologously expressing enzymes TPA dioxygenase (TPADO) and DCD dehydrogenase found in Comamonas sp. strain E6. For this step, we are proposing to use the following Biobricks from 2012 TU-Darmstadt team: K808011/ tphA1, k808012/ tphA2, and K808013/ tphA3 encoding TPADO converting TPA to DCD; K808010/ tphB and K808014/ AroY encoding DCD dehydrogenase converting DCD to PCA. Second, P. putida can convert PCA to lactate using its native metabolism and secrete lactate extracellularly via the lactate transporter. To increase lactate production, we could maximise the flux towards lactate production while maintaining P. putida’s biomass growth at a desired level. This can be achieved by, first, upregulating genes essential to TPA degradation pathway; second, deleting non-essential genes. FBA will provide us with a list of genes to upregulate and delete, but we are not conducting any wet lab to test this aspect.

S. oneidensis will uptake lactate secreted by P. putida as the only carbon source to support its biomass growth. S. oneidensis, being exoelectrogenic, can channel the electrons to the anode via different electron transfer mechanisms, such as direct electron transfer, transfer via electron transport shuttles, or via pili, some of which will be modelled using an agent-based model (ABM).

3. Which whole organisms, including viruses and cell lines, are you planning to use or using in your project? Please provide as much detail as possible (such as strain information). If you are not using an organism, please note this.

If we had access to the lab, we would have co-cultured three species of bacteria: Escherichia coli BL21, Pseudomonas putida KT2440, and Shewanella oneidensis MR-1.

We have not had the opportunity to work with these organisms in the lab. Therefore, our data is being generated in silico through FBA and cellular automata modelling to mimic co-culture in the anode chamber.

4. What risks could these organisms pose to you or your colleagues in the laboratory, or to your community or the environment if they escape the lab?If you are not using an organism, please note this.

Although our project will be dry lab based, we are aiming to inform the future use of the three bacterial strains for the purposes of the iGEM competition. Therefore, we simulated “on paper” the process of how we would culture these organisms, how plasmids with genes of interest would be expressed in these organisms, how the proteins would be purified, and how their activities would be tested. Apart from the ‘on paper’ work, we are collaborating with this year’s Exeter team, asking them to help us carry out enzyme activity analysis, mainly Para-nitrophenyl-acetate (pNP-acetate/pNPA) assays, Western Blots and High Performance Liquid Chromatography (HPLC). From the safety aspect, last year’s Exeter team

has experience in carrying out pNP- acetate assays and HPLCsafely and have created the necessary safety documentation. We have discussed the strains, plasmids and experimental plan with the Exeter team via virtual meetups between the UCL and Exeter teams including the supervisors. The teams are going to share each other’s iGEM safety forms and plan the step of the protocols together to ensure that the experiments are safe to the Exeter team. As the project is progressing, we will continue to update the safety forms accordingly.

Details regarding the potential risk posed by the three bacterial species are provided below:

E. coli is an opportunistic pathogen that commonly lives in the mammalian gut; however, this pathogenicity is usually dependent on the E. coli strain. The risk of pathogenicity of the laboratory E. coli strain BL21 is considered to be low. It is a non-colonizing and disabled strain that is considered not to carry the well-recognized pathogenic mechanisms required by strains of E. coli causing most enteric infections. BL21 is classified as ACDP (Advisory Committee of Dangerous Pathogens) hazard group 1. Thus, it is non-pathogenic and unlikely to survive in host tissues and cause disease. Although the strain is not considered pathogenic to humans or animals, there may be low health risks associated with irritation of the eye and skin, and mild respiratory tract inflammation if contacted, ingested or inhaled. In terms of biocontainment, the strains being used in the co-culture are expected to have limited survivability in the environment given that although wild type E. coli and P. putida consume substrates other than PET and TPA, respectively, they will be engineered to be auxotrophs, thereby depending on each other for survival and minimizing potential escape. In the unlikely event of genetic escape, the strains we are using might transfer our designed plasmids via horizontal gene transfer to other bacteria in the environment thereby allowing them to acquire antibiotic resistance.

P. putida also belongs to the biosafety level 1 group. Its pathogenicity is highly dependent on the strain. We will be using the non-pathogenic P. putida KT2440 since it is known to degrade PCA natively. Other species such as P. aeruginosa or P. syringae have also been reported as opportunistic human pathogens capable of causing nosocomial infections. Because of this risk, we chose the non-pathogenic P. putida as one of our chassis for the MFC. Finally, S. oneidensis is also classified as biosafety level 1 and is a non-pathogenic gram-negative microbe. Although S. oneidensis can uptake carbon sources other than lactate under both aerobic and anaerobic conditions and grow in temperature ranging from approximately 0~35°C, it’s escape should not cause much safety concerns as our project only works with a non-pathogenic wild type strain.

To minimize the risk of infection, gloves should always be used as well as adhering to good aseptic techniques when handling these strains under lab conditions. Alternatively, kill switches have been shown to be effective at ensuring containment which we could incorporate, for instance into P. putida. This strategy would involve the expression of a toxin in the absence of PET, thereby ensuring containment.

5. What organisms are you using as chassis in your project?For the purposes of iGEM, a chassis is the organism in which you are putting your parts, or which you are modifying in your project. Many teams will use a

common lab organism as a chassis. Some teams may use a more exotic organism. Some project may not involve a chassis.

Escherichia coli BL21

Pseudomonas putida KT2440

6. What risks could your chassis pose to you or your colleagues in the laboratory, or to your community or the environment if they escape the lab? If not using a chassis organism, please note this.

We are using E. coli BL21 DE3 and P. putida KT2440 as chassis. The following are the potential risks associated with:

1. Risk of co-culture: If the engineered cells are not robust enough, they could exchange DNA that allow them to co-evolve while acquiring new functions such as antibiotic resistance, thereby creating a potential risk of increased AMR that our current medical technologies cannot handle.

2. Risk of escaped GMOs surviving in nature: A solution to these problems is to make E. coli and P. putida Lys and Leu auxotrophs. This would make them dependent on the essential amino acids produced by the other bacterium. By engineering E. coli to be LysA- and P. putida to be LeuA-, we minimized the chance of escaped E. coli and P. putida surviving in nature.

3. Risk of genetic escape: Since the full PET degradation pathway is split between two different organisms, the chance of another microbe acquiring the entire pathway is reduced. Moreover, successful degradation of PET requires the native PCA degradation genes which are present in the chromosomal DNA of P. putida rather than the plasmid, thereby escape of the PCA degradation pathway is very unlikely.

7. Which chemicals are you using in your project that might be hazardous?

Heavy metals Carcinogens Mutagens Other controlled chemicals

Below is a list of chemicals that would be used if we had lab access and were carrying out experiments. We will not be using these chemicals ourselves, but some of these may be used by our collaboration partner (Exeter team). Please see question 29 for further details.

1. Para-nitrophenyl-acetate: stable, combustible, incompatible with strong oxidizing agents. May irritate eyes, skin, and respiratory tract.

2. Sodium phosphate buffer: Causes mild skin irritation. Material may be irritating to the mucous membranes and upper respiratory tract. May be harmful by inhalation, ingestion, or skin absorption. May cause eye and respiratory system irritation.

3. Sodium Chloride: Ingestion of this compound may cause nausea, vomiting, diarrhea, muscular twitching, inflammation of the gastrointestinal tract, dehydration and congestion (dehydration and congestion can occur in most internal organs, particularly the meninges and brain).

4. Formic acid 0.1%: Chronic Exposure: Prolonged exposure causes local irritation of skin and mucous membranes, especially to the eyes. No component of this product present at levels greater than or equal to 0.1% is identified as a known or anticipated carcinogen by NTP, IARC, or OSHA. Inhalation: Remove to fresh air

5. Acetonitrile: toxic, colorless liquid with an ether-like odor and a sweet, burnt taste. It is an extremely dangerous substance and must be handled with caution as it can cause severe health effects and/or death. Containers of the liquid can explode when heated.

6. Acrylamide: increased risk of developing cancer if ingested.

7. Tris-HCl buffer: May be harmful if inhaled. May cause respiratory tract irritation. May be harmful if absorbed through skin.

8. Sodium Dodecyl Sulfate (SDS): Causes respiratory tract irritation. Skin Toxic if absorbed through skin. Causes skin irritation. Eyes Causes eye irritation.

9. Ammonium persulfate: May intensify fire; oxidizer. Harmful if swallowed. Causes skin irritation. Causes serious eye irritation. May cause allergy or asthma symptoms or breathing difficulties if inhaled. May cause an allergic skin reaction. May cause respiratory irritation.

10. TEMED: Highly flammable vapor and liquid. Causes serious skin irritation. May cause severe digestive tract irritation with abdominal pain, nausea, vomiting and diarrhea. May be harmful if swallowed. May cause respiratory tract irritation. Irritation may lead to chemical pneumonitis and pulmonary edema.

11. Dithiothreitol: Causes skin irritation. Causes serious eye irritation. May cause respiratory irritation.

12. Glycerol: causes serious eye irritation.

13. Bromophenolblue: May cause irritation to the respiratory tract. Large oral doses may cause irritation to the gastrointestinal tract. May cause irritation to the skin. May cause irritation to the eyes.

14. PMSF: according to Harvard Campus Services - Environmental Health & Safety, it is labelled as "Toxic if swallowed" and "Extremely corrosive and destructive to tissues; May cause irreversible eye damage." Also, PMSF hydrolyzes upon exposure to water/moisture, liberating a toxic and corrosive gas (HF) that in contact with metal surfaces can generate flammable and/or explosive hydrogen gas.

15. TCEP: California State lists TCEP as a carcinogen. Studies also found that it can cause reproductive effects and neurotoxicity.

When handling any of these chemicals, PPE such as goggles, gloves and lab coats must be worn at all times, and handling must take place under a certified fume hood, working behind the sash.

8. As part of your project, are you are planning to make / have made new parts or substantively changed existing parts in the Registry.

Yes (All relevant new or revised parts should be described on a spreadsheet) No

9. See spreadsheet

10. What experiments will you do with your organisms and parts? Please explain briefly. We are particularly keen to understand the boundaries or scope of your project. You should include the names of species / cell lines / strains. You should include experiments involving parts taken from other

organisms, even if they are being synthesized rather than isolated from nature – you need not include any parts already in the registry.

Dry lab:

Our project is mainly dry lab focused and involves a high degree of modelling; however, Benchling has been used to design our constructs to be uploaded to the iGEM registry. We will create and test a novel PETase-MHETase fusion protein for the efficient degradation of PET to TPA in E. coli. The following 4 DNA constructs have been designed: (1) LamB-PETase-G4S-MHETase-His, (2) LamB-PETase-rigid linker-MHETase-His, (3) LamB-PETase-His and (4) LamB-PETase-His. Each of the PETase and MHETase genes are fused with a LamB secretion tag at the N terminus and a hexahistidine tag on the C terminus. PETase and MHETase are connected via a flexible (1) or rigid (2) polypeptide linker. Our genes will be synthesized by a company and cloned into an pSB1C3 expression vector that contains a T7 promoter along with a strong ribosome binding site based on part BBa_K3111102 and in line with iGEM cloning convention. We will test for secretion of the proteins of interest into the growth medium via the secretion tag LamB when expressed in E. coli. We are also proposing experiments for next year’s team to perform on P. putida, S. oneidensis, and MDC efficiency. We also had an idea for a biocontainment kill switch for P. putida, the switch would be controlled under an inducible promoter, that will only be activated upon addition of a compound that will restart the MFC in order to maintain its optimum performance.

Wet lab:

Wet lab work planned to be carried out by our collaborator Exeter 2020 team: Measuring expression, secretion and activity of our PETase-MHETase fusion protein.

These are the experiments being conducted by Exeter:

1. E. coli BL21(DE3) transformation and growth protocol:

Synthesized DNA (4 samples) will be shipped to UCL where a PhD student (from the iGEM supervisor’s lab at the Department of Biochemical Engineering) will prepare the DNA to be shipped to Exeter. The PhD student has had the required lab safety training and is an experienced postgraduate student and iGEM supervisor. At Exeter transformation into BL21(DE3) will be carried out and the strains will be cultured/genes expressed. In brief, a tube of BL21(DE3) Competent E. coli cells is thawed on ice. After gentle mixing, 50 µl of cells are pipetted into a transformation tube. 1–5 µl containing 10-100 ng of plasmid DNA is added to the cell mixture. The mixture is placed on ice for 30 minutes. Cells are heat shocked at exactly 42°C for exactly 10 seconds. Mixture is placed on ice for 5 minutes. 950 µl of room temperature LB growth media is pipetted in mixture. Tubes are then placed at 37°C for 60 minutes and shaken vigorously (250 rpm). 50–100 µl of each dilution is spread onto a selection plate (containing 35 mg/L chloramphenicol) and incubated overnight at 37°C.

Liquid LB Growth media with chloramphenicol (25 mg/L) is inoculated with colonies taken from the selection plates overnight. Cells are induced with IPTG and proteins are expected to be secreted via the SEC secretion machinery. Cells are centrifuged at 40,000 g and the supernatant is collected.

2. Protein purification:

A standard Nickel nitrilotriacetic acid (Ni-NTA) protein purification procedure is carried out to isolate Histidine-tagged proteins via affinity chromatography using a drip column. Protein-containing sample is loaded into the column after equilibration with Buffer A (containing 50mM Tris HCl at pH7.5, 0.5M NaCl and 20mM Imidazole). The unbound protein is washed out with Buffer A and the flow through is collected in

fractions of 10 ml. Buffer B (containing 50mM Tris HCl at pH 7.5, 0.5M NaCl and 500mM Imidazole) is then used to wash the column, eluting any bound protein; fractions of 2 ml are collected.

3. An SDS-PAGE assay is conducted to check for successful purification. Detailed protocols are provided by the University of Exeter’s 2020 iGEM team on their wiki. A risk assessment for this assay technique will also be provided by the team.

4. Measuring degradation of PET

To measure the degradation of PET, a fluorescent-based or calorimetric assay, for example the pNP-acetate assay, is a good way to measure the activity of our PETase. As the pNP acetate ester bond is cleaved, it generates nitrophenolate, causing an increase in absorbance at 405nm. The more activity our PETase has, the higher the absorbance that will be recorded. PNP-acetate assay with PETase alone will be conducted as positive control group while blank plasmid will be transformed into E. coli BL21 as negative control. HPLC or Mass spectrometry would be able to separate the products (MHET, TPA and EG), thereby facilitating their individual quantification and indicating the activity of MHETase.

4.1. PNP assay:

The following reagents are used: Para-nitrophenyl-acetate 50 mM, Sodium Phosphate (NaH2PO4) buffer pH7.5, 100 mM NaCl. A range of substrate concentrations (between 0-20 mM pNP-A) are tested and a blank is used to subtract the auto-hydrolysis of the pNPA. The production of p-nitrophenol is measured at 405 nm. The solutions are mixed together, and the substrate solution is added immediately before the spectrophotometer read is made.

4.2. Standard HPLC assay:

Samples (10 µl) are analysed using a high-performance liquid chromatography system (HPLC, Agilent 1200) using an Eclipse Plus C18 column (Agilent, UK). The mobile phase consisting of 99.9 % Water with 0.1 % Formic Acid is pumped at a flow rate of 0.8 ml min-1 and the effluent monitored at 240 nm. The typical elution condition is 10 min with 20% - 80% acetonitrile. The amounts of products (BHET, MHET and TPA) are calculated by comparison to a standard curve. All samples are analysed in triplicate and the data averaged and standard errors calculated.

Potential experiments for next year’s team: Measuring TPA uptake and lactate secretion in P. putida; measuring lactate concentration for S. oneidensis; measuring our MDC’s efficiency.

To measure the uptake of TPA by P.putida after expressing the exogenous TPA transporter, absorbance measurements of cell suspensions could be taken over time. A decrease in TPA concentration would indicate uptake of TPA by P. putida.

Regarding S. oneidensis MR-1, uptake of lactate would need to be measured to verify if overexpression of its lactate transporter correlates with the higher voltage generated, we would need to measure the lactate concentration over time. A decrease in lactate concentration would indicate that S. oneidensis is successfully taking up lactate. The voltage generated by the MFC can be measured with a voltmeter.

We will also measure desalination in the anode chamber and report the decrease in salt concentration when compared to the original seawater, thereby we can measure the efficiency of our device.

Desalination can be measured using an electrical conductivity (EC) meter since conductivity correlates with the salt concentration.

11. What risks could arise from these experiments?For example, could they produce aerosols making it more likely that you could inhale something? Or are you using needles and could accidentally stick yourself? Could you produce something that is not inactivated using standard lab protocols? If you are not conducting any experiments, please note this.

We are not conducting any experiments ourselves at UCL.

There are 3 types of potential risks that should always be considered in a lab environment when team Exeter is performing experiments for us: biohazards, chemical hazards and physical hazards.

1.Biohazards (please refer to Question 4):

If microorganisms are accidentally released into the environment during the process of the experiments:

1.1 E. coli BL21 (DE3):

-Risk: Biosafety level 1. Low risks of gut infection and colonization, spills since it is considered not to be pathogenic. However, chassis of E. coli BL21 might cause human safety, environmental, ecological issues if released.

2.Chemical hazards (please refer to Question 7):

2.1 NaCl and HCl

-Risk: Skin irritation and inhalation of chlorine gas present in the middle chamber.

-Prevention/Protection: Wear PPE and avoid spills on the skin.

2.2 Terephthalic Acid (TPA)

-Risk: can affect you when breathed in. Contact can irritate the skin and eyes. Breathing it can irritate the nose, throat and lungs causing coughing, wheezing and/or shortness of breath. Repeated exposure may affect the kidneys.

-Prevention/Protection: Wear PPE and avoid inhalation of the chemical.

2.3 Mono (2-hydroxyethly) terephthalate (MHET)

-Risk: Toxic chemical similar to BHET. It could cause injury mainly in livers and kidneys. Moreover, with the benzoate structure, it can also have some toxicity from benzoate.

-Prevention/Protection: Direct contact should be avoided, PPEs are needed.

2.4 Bis (2-hydroxyethly) terephthalate (BHET)

-Risk: Experiments conducted on non-human subjects have shown that BHET is toxic due to its ability to increase the activities of a reductase system. However, since it contains benzoate in its structure, some safety issues of benzoate might be inherited.

2.5 Ethylene glycol (EG)

-Risk: Moderately toxic. Upon ingestion, EG is oxidized to glycolic acid, which is, in turn, oxidized to oxalic acid, which is toxic. It and its toxic byproducts first affect the central nervous system, then the heart, and finally the kidneys. Ingestion of sufficient amounts is fatal if untreated. EG can be broken down in air in about 10 days and in water or soil in about a few weeks. Prolonged low doses of EG show no toxicity, at near lethal doses (≥ 1000 mg/kg per day). EG acts as a teratogen.

3. Physical hazards:

3.1 Broken glass:

-Risk: Skin cuts

-Prevention/Protection: Wear gloves and throw remains in the broken glassware bin.

3.2 Needles:

-Risk: Sticking fingers.

-Prevention/Protection: Wear gloves and handle them with care.

3.3 Spills:

-Risk: Skin irritation

-Prevention/Protection: Mop up the spill with an adsorbent material.

3.4 Fire:

-Risk: Skin burns

-Prevention/Protection: Ring fire alarm and exit building while ensuring all fire doors are kept closed to prevent fire spread.

3.5 Malfunctioning electrical equipment:

-Risk: Electric shocks

-Prevention/Protection: Verify the state of all wires. Turn off the switch of any electrical equipment after using it as a good laboratory practice.

12. Are you collecting any data about people, such as their opinions, quotations, health information, gender, behavior, attitudes, or concerns?This includes surveys and interviews carried out as part of human practices work, whether anonymous or not.

No Yes (Please read iGEM's policy on Human Subjects Research very carefully. For

good reasons, many countries require formal approval for Human Subjects Research, as well as consent procedures for participants. You may need formal permission from a Research Ethics Committee, Institutional Research Board, or equivalent. Remember

compliance with relevant laws and regulations is a requirement for participation in iGEM.)

13. Imagine that your project was fully developed into a real product that real people could use. How would people use it?

Our project is foundational / we do not have a specific real-world application in mind (Examples: library of standardized promoters, system for communication between cells)

Only in the lab (Examples: reporter strain for measuring the strength of promoters)

In a factory (Examples: cells that make a flavor chemical for food, cells that make biofuel)

In a consumer product that ordinary people buy (Examples: cells that clean your clothes, bread made with engineered yeast)

In agriculture / on a farm (Examples: cells that guard against pests, engineered rice plants, cells that promote growth of crop plants)

In a small enclosed device (Examples: a bio-sensing strip with cells that detect arsenic)

In the natural environment (Examples: cells that remove pollution from lakes, engineered forest trees that can resist drought)

To be used in the human body, or in food (Examples: anti-cancer bacteria, bread made with engineered yeast, engineered rice plants)

Other (Examples: bacteria that live on Mars, or a software project)

14. What safety, security or ethical risks would be involved with such a use?Virtually all modern life science and biotechnology carries with it some risks. These can be identified and managed helping to ensure your work makes a positive impact on the world. Basic risks are managed for you in many institutions. As you think about how your project might enter the real world, being a responsible biological engineer will require you to think about and manage these risks yourselves. You can find some great resources on the Safety and Security and Human Practices hubs on our wiki. It is even possible that software projects could also pose relevant risks.

1.Pre-implementation (Risks of conducting market research): We have considered safety and security risks of the population in the test and target market due to data collection and analysis during market research. Market research has already been initiated as one of our Human Practices activities: We

choose California as our project's test market while Nigeria and Ghana as our target market because all these regions are suffering from oceanic plastic pollution and water scarcity.

1.1 Risks of interview:

We have conducted interviews with two people from our two proposed target markets respectively. Full consent was given by both interviewees including recording the interview by the interviewer. When designing interview questions, we have looked upon resources shared on iGEM Human Practices Hub, especially Harvard: Strategies for Quantitative Interviews for interview questions and protocol design.

1.1.1 Interview with a farmer from Ghana whose contact details were shared by team Ashesi

1.1.2 Interview with the Executive Director from "RecyclePoints", an NGO aiming at tackling plastic pollution

1.2 Risks of conducting surveys:

We have conducted two surveys. Questions in both surveys were designed in accordance with the guidance provided on the Human Practices resources of iGEM (Johnny, B. et al. (2013). Designing Surveys 3rd edition). To ensure we comply with General Data Protection Regulation (GDPR), we designed our surveys based on collecting as little personal information as possible. We informed our readers at the beginning of our survey about how the results would be analysed with our promise of not illegally sharing their data to enhance transparency. We ensure only adequate, accurate data would be analysed. Finally, each survey template was triple reviewed by our team supervisors, team members, and people or organizations who helped us distribute the surveys (i.e. Ashesi team members and supervisors and directors of RecyclePoints). We received guidance from an expert in particular at UCL, Dr Elpida Makrygianni.

1.2.1 A joint survey collaborating with Ghana Ashesi team

1.2.2 Our own survey which was helped by a Nigerian NGO "RecyclePoints" for distributing

2.During Implementation (this stage includes risks associated with construction and operation of desalination plants):

2.1. Risks of constructing and operating plants in coastal regions in test and target markets.

2.1.1 Ethical risks to the local community where residents may have concerns regarding the construction and operation of plants consisting of GMOs. Also, the construction can also force residents to move away which they may not be willing to.

2.1.2 Risks to construction workers as well as workers operating the MFCs in the plants.

We have also gone through some well-known companies' staff safety manuals so that not only do we adhere to, but also know how to ensure staff safety.

2.2 Risks regarding the quality and safety of water for irrigation

2.2.1 Although the risks of the desalinated water being unsafe is low, we recognized that the desalinated water would need to undergo regulations regarding water quality and be further treated and disinfected before being used for irrigation purposes. Thus, we have conducted a visit to Shanghai VEOLIA Linjiang Water Treatment Plant to make it clearer for us how water is supplied from origin to end users in the real world. The visit was started with the video of visitors' safety instruction and helmets and reflective vests were on during the whole visit.

2.2.2 In order to minimize the potential risks of people using unsafe water, monitoring systems must be in place all the time and consumers should be informed of whether the crops are irrigated with GMOs involved desalinated water or water from other sources to ensure we do not pose ethical issues.

2.3 Risks regarding exhaust and brines from desalination plants

2.3.1 Chlorine and hydrogen gas might be the by-product of desalination using electricity which are toxic and flammable. However, according to a paper written by David from MIT News Office, these two gases are the raw materials for making hydrochloric acid which can be recycled for the process of desalination to help clean parts of desalination plants

2.3.2 Sodium hydroxide could be a potential by-product of the cell and result in a potential risk to the environment if released by accident. However, again as for 3.1, sodium hydroxide from desalination brines can also be recycled for sea water pre-treatment and dealing with membrane fouling by changing acidity of the water.

2.3.3 Despite that brines can be recycled; the recycling technologies are not mature. Therefore, in this current stage, brines from desalination must be diluted before pumped back to the ocean. This ensures ethical risks of damaging the ecosystem of marine lives are minimized.

2.3.4 Waste disposal from cell operation. The cell debris and metabolites accumulated in the anode chamber would need to be disposed safely.

15. How will experts overseeing your project help to manage any of the risks you identified in this form?

The team is working closely with Dr Brian O'Sullivan, the Departmental Safety Officer of UCL Biochemical Engineering. Brian has many years of experience in working with previous UCL iGEM teams. Unfortunately, due to COVID-19, our team has not been able to get into the lab this summer, and for the duration of the iGEM project. Therefore, we will not be exposed to many of the risks identified above as we will not be the ones conducting experiments. As detailed in Question 10 and 29, Exeter’s iGEM 2020 team will be doing some experiments for us, but we have still included in this form all the risk management strategies that they will be implementing when conducting experiments for us. Risk management is being facilitated by the following:

1.Brian will be reviewing our safety hazard forms for every experiment and ensures that we are following safety guidelines and protocols. Our academic supervisors, Dr. Stefanie Frank and Dr. Kenth Gustafson also have extensive experience in research and advise us regarding safe working practice.

2.We have familiarised ourselves with UCL’s safety regulations if we were to ever enter the lab, including submission of a Risk Assessment form for every experiment. The Risk Assessment form lists possible hazards from the activity, and the control measures required. It would be signed off by our

academic supervisors and the departmental safety officer, who checks that the control measures are in place. Link to UCL Safety Handbook:https://www.ucl.ac.uk/safety-services/a-z.

3.Overall supervision: The Departmental Safety Officer at the Department of Biochemical Engineering provides safety training and inductions, organizes regular lab inspections, administers Safety Committee meetings, manages risk assessments, updates safety policy, and ensures the department is compliant with legal requirements and UCL safety regulations.

4.Communications with experts in companies have also help us to identify some problems that we might face in real-world applications such as waste disposal, regulations of water quality for irrigation, construction of plants in coastal regions.

16. What rules or guidance cover your work which could help to manage any of the risks you identified in Part 2 of this form (in particular Question 10)? For example: In your country / region, what are the laws and regulations that govern biosafety or biosecurity in research laboratories? Please give a link to these regulations, or briefly describe them if you cannot give a link. What are the guidelines for laboratory biosafety and biosecurity? Please give a link to these guidelines, or briefly describe them if you cannot give a link.

The UK's Health & Safety Executive provide relevant information on the subject of GMO containment (https://www.hse.gov.uk/biosafety/gmo/index.htm). We also accessed the most recent GMO regulations from 2014 for further information: https://www.hse.gov.uk/pubns/books/l29.htm. Regarding safety concerns at our institution, UCL has mandatory requirements when working with GMOs, including the use of an online risk assessment tool in RiskNet (https://www.ucl.ac.uk/safety-services/risknet). This helps departments at UCL manage their safety responsibilities and we will strongly consider using this tool in next year’s wet lab context. UCL has also put together a Biosafety Design Initiative (https://www.ucl.ac.uk/biosafety-design-initiative/), which we could contact to learn more about biocontainment and get expert advice.

17. Have your team members received any safety training?For the purposes of iGEM, biosafety and biosecurity training covers the procedures and practices used to manage risks from accidents or deliberate misuse of your projects to your team, colleagues, communities and the environment. All team members are expected to be aware of these risks and to work to manage them.

Yes, we have already received safety training. Yes, we have already received security training. We plan to receive safety and security training in the future. Please specify

approximately when:

We will not have safety and security training. Please explain in detail how team members will be aware of and manage risks to the team, colleagues, communities, and the environment in the absence of training. If it is not relevant because there is no lab component to your project, please note this:

All team members have received general UCL safety training on their course; however, as there is no wet-lab aspect in our project this year, we will not have the specific safety and security training that is given to iGEM team members every year at UCL. To gain awareness of risks and how to manage them, multiple team members attended a Risk workshop on biosafety and biosecurity by the iGEM safety committee. Through this, we were able to identify the safety and security risks in different scenarios and actions to mitigate them. Additionally, this safety form was reviewed by the departmental safety officer, Brian O’Sullivan.

We will be performing experiments in partnership with Team Exeter though we will not be handling DNA by ourselves. All constructs will be prepared by a PhD student with appropriate lab training.

18. Please select the topics that you learned about (or will learn about) in your safety training.

Lab access and rules (including appropriate clothing, eating and drinking, etc. Responsible individuals (such as lab or departmental specialist or institutional

biosafety officer) Differences between biosafety levels Biosafety equipment (such as biosafety cabinets) Good microbial technique (such as lab practices) Disinfection and sterilization Emergency procedures Transport rules Physical biosecurity Personnel biosecurity Dual-use and experiments of concern Data biosecurity Chemicals, fire and electrical safety We will not have safety training

19. Which work areas do you use / are you using to handle biological materials? Please check all the containment provisions you are using. If you are using more than one space please check all that apply and note this in the other work area box.

No lab work (e.g. software project) Open bench Biosafety cabinet (please note there are important differences between

biosafety cabinets and laminar flow hoods / clean benches. iGEM encourages the use of biosafety cabinets but discourages the use of laminar flow hoods or clean benches. This Factsheet from the University of Massachusetts Amherst helps explain the differences.)

Specialist greenhouse Specialist animal house Specialist insect facility Chemical fume hood (Please note – this is designed to manage risks from

hazardous chemicals. It is different from a biological safety cabinet designed to manage risks from hazardous biological agents and a clean bench or laminar flow hood designed to prevent contamination of your experiment.)

Other work area. Please describe: Unknown. Please comment: Our lab work will be performed by Exeter iGEM team

and therefore please refer to their safety form for details on their work area.

20. What is the biosafety Level of your work space?

Not applicable as we have no lab component We have several different lab spaces with different biosafety Levels. Please

describe:

BUT – Exeter iGEM 2020 are helping our project by conducting a number of experiments (detailed protocols are attached to Q10 of this form)

In their work for us, the biosafety level of their workspace will be level 1 (low risk)

Level 1 (low risk) Level 2 (moderate risk) Level 3 (high risk) Level 4 (extreme risk) Other biosafety level. Please describe:

21. What other risk management tools will cover you work?

Accident reporting (measures to record any accidents) Personal Protective Equipment (including lab coats, gloves, eye protection, etc)

An inventory control system (measures to track who has what materials and where they are)

Access controls (measures to control who can access your work spaces, or where materials are kept)

Medical surveillance (measures to find out if you get sick because of something you were using)

Waste management system (measures to make sure waste is not hazardous before it leaves your institution)

Special procedures or protocols that address safety or security Others Please describe:

22. How will the rules, training, containment and other procedures and practices help to manage any of the risks you identified in Part 2 of this form (in particular Question 10)?Please give details of any steps you have taken to manage any risks identified. This might include how any of the following have helped manage risks: the rules you identified, the training you have had, the equipment you have used, the spaces you have worked in, and the procedures and protocols you have followed. It might also include things you deliberately didn't do. For example, if you are not conducting any experiments, especially on grounds of safety, security or as a responsible scientist / engineer, please note this. Examples might include, making sure you only use non-pathogenic strains of an organism, deciding that animal use experiments are not yet warranted, or avoiding plant infection experiments because the affected plant is found in your country. Please also consider waste treatment – how will you know that any waste produced in your project will be successfully inactivated?

Due to the pandemic situation in London, we are not conducting any wet lab experiments this year; however, the training received from experts mentioned in Question 15 and 17 have helped us to be aware of how to minimize biosafety-related risks in the lab as well as all other risks mentioned when working in a wet lab environment. The rules identified in Question 16 have given us guidance on how to proceed safely when using GMOs.

1.In response to the risks set out in Question 11, containment strategies will be used to limit the growth of E. coli outside the co-culture environment:

1.1 A non-pathogenic strain BL21 DE3 is used to minimise risks.

1.2 Growth can be controlled with a toggle switch or an inducible promoter capable of switching off the transcription of PETase and MHETase, thereby limiting E. coli from feeding on PET, consequently reducing its growth.

2.For the Exeter team who will be conducting experiments on E. coli for us, the following risk management strategies are being implemented to prevent risks due to biological, chemical and physical hazards:

2.1 Personal protective equipment (gloves, lab coats and goggles) should be worn in the lab at all times.

2.2 In case of spills on skin or contact with the eye, rinse thoroughly with water for 15 minutes

2.3 Broken glass should be thrown in the broken glassware bin.

2.4 Needles should be handled with care and disposed into a sharps disposal container to prevent harm.

2.5 Spills on the work surface should be cleaned up immediately with an adsorbent material.

2.6 In the case of a fire, ring the fire alarm and exit building while ensuring all fire doors are kept closed to prevent fire spread.

2.7 Lab equipment should be checked routinely, appliances with exposed wires should not be used. Switch off any electrical equipment after use to minimise energy use.

3.In terms of the ethical risks that arise from this project and described in Question 14, we have been given adequate training to record such data and are following iGEM’s human practices safety policies (please see Question 29 for further details).

23. Are you planning to/ have released any organism or product derived from your project? For the purposes of iGEM, release includes putting any engineered organism or product from one into the environment, yourselves or volunteers (including by eating or drinking), or into a device that will be placed in the environment.

No Yes

STOP: Release is not allowed in iGEM. For more information see the policy page. Please contact the Safety and Security Committee by emailing safety AT igem DOT org

24. Are you planning to use, or using any animals (including insects and invertebrates) not on the Whitelist?

No Yes

STOP: Before you acquire or use any animal NOT on the Whitelist, you must - submit a

Check-In form. Check-Ins allow the iGEM Safety and Security Committee to help you ensure that you will work safely and responsibly with these organisms. The Safety and Security Committee will base its review on the information you provide – please provide as much information as possible and use references as appropriate.

25. Are you planning to use / have used any vertebrates (e.g. rats, mice, guinea pigs, hamsters) or higher order invertebrates (e.g. cuttlefish, octopus, squid, lobster)?

No Yes

STOP: Before you acquire or use any vertebrate you must submit an Animal Use form. The use of animals is not allowed in iGEM projects without a special exception from the Safety and Security Committee. For more information see the policy page and the White List. Please contact the Safety and Security Committee by emailing safety AT igem DOT org

26. Are you planning to use, or using any parts not on the Whitelist?

No Yes

STOP: Before you acquire or use any part that is NOT on the Whitelist, you must - submit a Check-In form. Check-Ins allow the iGEM Safety and Security Committee to help you ensure that you will work safely with these riskier parts. The Safety and Security Committee will base its review on the information you provide – please provide as much information as possible and use references as appropriate.

27. Are you planning to carry out any of the activities not on the Whitelist?These include experiments likely to bias the inheritance frequency of a genetic marker in an organism’s progeny, such as through the creation of a gene drive, experiments likely to confer resistance to the World Health Organization's list of Critically Important Antimicrobials, and experiments likely to increase the hazard posed by your project.

No Yes

STOP: Before you carry out any of the activities NOT on the White List, you must -

submit a Check-In form. Check-Ins allow the iGEM Safety and Security Committee to help you ensure that you will work safely with these riskier organisms/parts. The Safety and Security Committee will base its review on the information you provide – please provide as much information as possible and use references as appropriate.

28. Are you planning to use, or using any parts or organisms obtained from outside the lab or regular suppliers?

No Yes

STOP: Before you acquire or use any organism/part that has come from outside the lab or regular suppliers, you must - submit a Check-In form. Check-Ins allow the iGEM Safety and Security Committee to help you ensure that you will work safely with these riskier organisms/parts. The Safety and Security Committee will base its review on the information you provide – please provide as much information as possible and use references as appropriate.

29. What else can you tell us about any risks associated with your project, how you are managing them, or your compliance with iGEM’s safety and security rules and policies? 

1. Other potential risks associated with our project:

1.1 This technology could be giving the public excuses to neglect plastic pollution as they might overestimate the plastic degrading capacity of our microbial desalination cells. If public perception leads to more plastic waste in the ocean than what our plants are capable of degrading, the ocean plastic pollution situation will face the risk of further exacerbation.

1.1.1 To manage this, we will make sure as a team to address clearly the plastic degrading capacity of our plants and continue raising public awareness on plastic pollution.

1.2 In addition to the risk of releasing biological organisms into the environment, our project could also have a risk of polluting the environment with the disposals of a microbial fuel cell.

1.2.1 If a prototype is made, it hopefully would not need to be disposed of and can be stored and used appropriately. Appropriate use includes proper maintenance and regular cleaning of the prototype, using safe and efficient bacteria-removal products. If we decide to dispose of it, rules and guidelines will be followed to avoid any harm to our environment.

1.3 We have conducted surveys collecting sensitive personal data and public opinions. Risks with processing the data include privacy concerns with ensuring secure data handling and maintaining confidentiality. The survey collecting personal data will include participants under 18 and with

disabilities. Therefore, there are risks with involving vulnerable groups and large-scale processing of special category data.

1.3.1 To manage these risks, we've applied for approval from our institutional data protection office and ethics review board. Data collection will be anonymous to protect the privacy of the participants. Participants will be provided with an information sheet and a consent form prior to data collection. Therefore, processing will only be performed based on informed consent. Additionally, the data collectors will go through a data protection online training provided by our institution. UCL education experts have also given us guidance on how to address minors, while ensuring everyone’s safety online.

1.3.2 All human subjects research will be compliant with iGEM's policies and institutional rules to make the research as ethical as possible. Multiple communications with iGEM’s diversity and inclusion committee have been made to ensure the respect of these policies.

1.4 There are additional risks associated with an on-line, software-based project to do with at-home sedentary work for long hours, including:

-Manual handling and upper limb disorders-Looking at computer screens for long hours-Slips, trips and falls-Stress-Fatigue + headaches-Electrical equipment.-Disruption of sleeping patterns (due to time-zone differences and location of team members)

-Fire risks

To mitigate these, we are implementing the following strategies:

1.4.1 Ensuring team members or supervisors are aware that manual handling must be done with a straight back, and that any upper limb disorders are run by the safety officer for our Biochemical Engineering Department to minimise the effect on the person (although this didn’t apply to our team).

1.4.2 Ensuring team members know to balance long hours of looking at screens (unnatural light) by going outside and looking at natural light

1.4.3 Ensuring team members know to keep their workspace clean and tidy so as to minimize fire-hazards and risks associated with electrical equipment.

1.4.4 Ensuring team members are not over-worked and that the workload is well distributed among team members.

1.4.5 Ensuring team members know to keep hydrated and to rest if they are feeling considerably unwell

1.4.6 Ensuring team members know not to connect too many extension cables together, and to not keep electronics near water

1.4.7 Ensuring team meetings are carried out at times that suit all team members, and that do not interfere with normal sleeping patterns

1.4.8 Advise team members to have working fire alarms within their home

1.5 There is an additional set of risks associated with working during the ongoing COVID-19 Pandemic. These include the risk of infection, as well as placing others at risk of infection.

1.5.1 To limit these risks, participants in the project were frequently reminded to wash their hands often with soap for at least 20s and to keep their social contacts to a minimum. Additionally, we advise team members to avoid crowded places and always wear a face mask if social contact is to be made.

2. Recap of iGEM's policies

2.1 Do not release policy- we will not be releasing our genetically engineered product into the environment

2.2 No human experimentation-our project is focused on the environment; therefore, we will not be using human samples in any experiments and will not test our product on humans.

2.3 Human subjects research- any surveys or other platforms that we will be using to reach out to the public will be sent preemptively to our public engagement experts at UCL, Dr. Elpida Makrygianni and Kim Morgan. In addition, as mentioned in question 14, our survey questions are designed fully based on the guidance in the resources provided by iGEM Human Practices hub while the templates of the surveys are triple reviewed. Data collected for analysis are ensured to be accurate and adequate only to follow GDPR compliance. As a UK team overall, we will strive to comply with the UK Research Integrity Office policies when including the public's input to our project.

2.4 No gene drives- We will not propagate genes or alter the probability of an allele to be inherited in the next generation of cells.

2.5 Anti-microbial resistance- Any anti-microbial resistance sequences will be specifically engineered in the cell types that need to be grown to ensure the success of our project. All these sequences will be reported as genetic modification and as a possible BioBrick if required. Not AMR-related sequences will be used for other microorganisms other than E. coli, P. putida and S. oneidensis.

2.6 Use of animals- our project does not require the use of animals.

2.7 Deletions as modifications- any deletions made to the sequences that we will be modifying will be considered a genetic modification by our team, in compliance with the iGEM policies.