Introduction

Ever since its inception, one of the core values of the iGEM competition is to use synthetic biology in a safe and responsible way. No matter the project, all iGEM teams should have a clear understanding of the risks associated with what they want to engineer and the potential implication of that technology once it is made available to the whole world. With this philosophy in mind, every aspect of our project, especially the wet lab, has been carefully assessed to ensure that our project will pose no harm and, if replicated, can be used safely by other scientists. As such, the safety page has been divided into four sections: the first three give more details about our training in general lab safety, as well as how we chose the organism we want to genetically modify and the skin protein target for the e-pili, while the fourth explores the data security procedures for our Human Practices.

General Laboratory Safety

Before starting the work in the lab, all team members, including those not being directly part of the wet lab aspect of the project, were trained with the proper standard safety procedures at the Manchester Institute of Biotechnology (MIB). All of the laboratories in the MIB are Biosafety Level-1, but some equipment needs special training before it may be used. Training in standard laboratory procedures, such as bacterial aseptic technique, was provided.

Standard personal protective equipment (PPE) was worn at all times when entering the lab. The PPE consisted of:

• EN374-3 compliant gloves (nitrile);
• EN166 compliant eye protection (chemical splash proof safety glasses);
• Howie laboratory coat.

Any possibility for cuts, abrasions and accidental needle stick/sharps injury were considered and carefully monitored. MIB waste disposal procedures were followed at all times. Biologically contaminated solid waste (e.g. agar plates, tips, gloves, pipettes etc.) was placed in biohazard waste bags and disposed of by MIB staff. Agar plates were placed within a separate lidded box and disposed of by MIB staff. Contaminated liquid waste was treated with Virkon at a final concentration of 1% for a minimum of 1 hour before disposing down the sink. Standard disinfection procedures (e.g. 1% Virkon or 70% ethanol) were followed.

We have also submitted a White list Check-in form to verify with the iGEM safety body that any chemicals and procedures we are performing satisfy iGEM’s criteria regarding potential hazards.

Type IV Pili Expression

In the early stages of our project, one of our main challenges was trying to understand if Geobacter sulfurreducens pili could potentially become a virulence factor, as type IV pili are known to mediate the pathogenicity of a number of different bacteria, e.g., Pseudomonas aeruginosa and enterohemorrhagic E. coli (EHEC)[1]. After extensive research and listening to the concerns raised by stakeholders (Manchester Institute of Innovation Research, The British Association of Prosthetics and Orthotics), we constructed a phylogenetic tree to determine the evolutionary distance between the pili of G. sulfurreducens and the pili of pathogenic bacteria. (See Model for more information).

Figure 1. Phylogenetic tree based on sequence divergence of pilin proteins closely related to G. sulfurreducens and P. aeruginosa, inferred from scraped BLAST data. The tree displays a distant relationship between G. sulfurreducens and any pathogen within the entire dataset.

Furthermore, our extensive dry lab experiments also unveiled significant structural differences between the pili of G. sulfurreducens and that of pathogenic bacteria such as P. aeruginosa, meaning that the pili we intended to work with likely lacked the means to mediate pathogenicity. G. sulfurreducens is a bacterium that lives in the soil and uses its pili to mediate iron reduction as part of its metabolism[2], and no research so far has linked it to any form of disease. Secondly, we had to select a suitable chassis organism for expressing the e-pili, since G. sulfurreducens is an anaerobic bacterium that requires more sophisticated growth conditions[3].

One potential candidate was Vibrio natriegens (used by the Tongji-China 2023 iGEM team), but its very close relationship to pathogens such as V. cholera or V. parahaemolyticus, as well as its classification as a BSL-2 organism in the United Kingdom, have deterred us from choosing it for our experiments [4,5]. P. aeruginosa, another bacterium that has the necessary machinery for the expression of type IV pili, was also not an ideal candidate due to its hazard to human health, having the potential of causing life-threatening infections [6]. After ruling out these organisms, we investigated whether E. coli could be used to express the pili we desire. While most E. coli strains do not express type IV pili, enterohemorrhagic E. coli (EHEC) do express the required machinery for their assembly [7]. The procedure from the paper "An Escherichia coli Chassis for Production of Electrically Conductive Protein Nanowires" by Ueki et al 2020. involved cloning the genes for the assembly machinery from EHEC into a non-pathogenic lab strain (NEB 10-beta)[8]. It is important to note that the assembly machinery itself is not pathogenic, but merely used to facilitate the assembly of the EHEC major pilin (PpdD) subunit into the pili polymer, which subsequently aids in adherence to host epithelial cells. [7] However, we wished to create a project that did not involve any BSL-2 associated gene or part. As such, we have looked at projects and papers that used alternative machineries for the pili production.

The Links China 2020 iGEM team amplified the transcriptionally inactive type IV pili assembly genes from the E. coli DH5 alpha genome and achieved expression of the G. sulfurreducens e-pili. Because one of the genes in the pili assembly system, encoding the prepilin-peptidase gspO, can only be found in E. coli K12 str. MG1655, we have decided to amplify the remaining components of the assembly machinery from the same strain. The proteins involved in the assembly machinery for the pili in EHEC and E. coli K12 share high homology, suggesting that the genes would most likely be functional once cloned. Afterwards, another challenge we encountered was the presence of the gene coding for the EHEC major pilin PpdD in the lab strain NEB 5-alpha, the strain which we used. We wondered whether expressing the assembly machinery in E.coli DH5alpha would increase the risk of the virulent pili being produced.

However, ppdD, as well as the other machinery genes found in E.coli K12 str. MG1655 and, by extension, NEB 5-alpha (since NEB 5-alpha is a lab strain derived from K12) are under tight transcriptional control and do not express the genes at any significant level[9]. Thus, the machinery we introduce will not pose any risk of creating ‘’harmful E. coli’’.

Choosing Collagen and Improving the Safety of the Collagen-Binding Assay

Our project aim was to engineer the G. Sulfurreducens pili to adhere to the surface of the skin, minimising the motion artefacts often experienced using myoelectric prosthetics. We realise that attempting to create any protein that interacts with the human physiology inherently possesses its own set of risks. After thorough research on potential binding candidates (e.g. integrin, keratin), we have decided that collagen would be the best choice for our proof-of-concept. Since collagen is not found on the outer surface of the skin, but rather in the dermis, the risk of accidental interactions with our skin would be minimal [10].

We aimed to test binding of our recombinant nanowires to collagen using a collagen binding assay protocol adapted from the paper "Collagen-binding activity of Prevotella intermedia measured by a microtitre plate adherence assay" by Grenier (1996) [11]. The original protocol used glutaraldehyde and crystal violet staining to fixate the cells to the collagen plates and measure the absorbance signals in the wells. Glutaraldehyde is known to be a toxic substance, so we improved the safety of the protocol by replacing this step. Instead, we equipped the cells with a fluorescent protein, encoded by plasmid pBbS1a-RFP and quantified adherence of the cells to the plate by measuring the fluorescence signal emitted by the cells [12].

Human Practices Data Security

In the UK, data protection is primarily governed by the UK General Data Protection Regulations (UK GDPR) and the Data Protection Act 2018[13]. These laws establish a comprehensive framework for processing personal data and ensure individuals’ rights regarding their information are respected. Key principles include the need for a lawful basis for processing, meaning that personal data must be processed lawfully, fairly, and transparently. Organisations must establish a legal basis for processing data, such as obtaining consent from individuals or demonstrating a legitimate interest. Data minimisation is another critical principle, which states that only the data necessary for the intended purpose should be collected and processed. Additionally, the purpose limitation principle mandates that data collected for one purpose can not be used for unrelated purposes without explicit consent. Individuals also possess rights over their data, including access, rectification, erasure, and restriction of processing. Finally, organisations must demonstrate accountability and governance by showing compliance with these data protection principles and implementing policies to safeguard personal data.

Having our team sign a consent form before they engaged in reflexive diaries was crucial, particularly when their analysed data is used on our team wiki. Consent forms ensure that the team members were informed about what data was being collected, how it will be used, and the implications of their participation. This practice aligns with the principles of lawful processing and transparency outlined in the UK GDPR. Furthermore, consent forms gave our team autonomy by allowing us to maintain control over our personal information. By explicitly agreeing to the processing of our data, we ensured that our rights were upheld, which was vital in fostering a positive research environment.

Additionally, a signed consent form provides documented proof that the team agreed to the use of their data, which is particularly important in collaborative projects where data sharing can be sensitive. Moreover, failing to secure consent can lead to non-compliance issues and potential penalties. Lastly, knowing that our data was used transparently and with our consent encouraged our team to participate more openly in reflexive diaries, ultimately leading to richer data and more insightful outcomes. Overall, adhering to data protection laws and ensuring informed consent is essential for safeguarding individuals’ rights and promoting a culture of trust within our iGEM team.

References

• Luna Rico A, Zheng W, Petiot N, Egelman EH, Francetic O. Functional reconstitution of the type IVa pilus assembly system from enterohaemorrhagic Escherichia coli. Molecular Microbiology. 2019 Jan 21;111(3):732–49.
• Howley E, Ki D, Krajmalnik-Brown R, Torres CI. Geobacter sulfurreducens’ unique metabolism results in cells with a high iron and lipid content. bioRxiv (Cold Spring Harbor Laboratory). 2022 Jul 30.
• Engel CEA, Vorländer D, Biedendieck R, Krull R, Dohnt K. Quantification of microaerobic growth of Geobacter sulfurreducens. Appanna VD, editor. PLOS ONE. 2020 Jan 16;15(1):e0215341.
• Hoff J, Daniel B, Stukenberg D, Thuronyi BW, Waldminghaus T, Fritz G. Vibrio natriegens: an ultrafast‐growing marine bacterium as emerging synthetic biology chassis. Environmental Microbiology. 2020 Jul 16;22(10).
• Vibrio natriegens [Internet]. Culturecollections.org.uk. 2022 [cited 2024 Sep 28]. Available from: https://www.culturecollections.org.uk/nop/product/vibrio-natriegens
• Fazeli H, Akbari R, Moghim S, Narimani T, Arabestani MR, Ghoddousi AR. Pseudomonas aeruginosa infections in patients, hospital means, and personnel’s specimens. Journal of Research in Medical Sciences: the official journal of Isfahan University of Medical Sciences. 2012;17(4):332–7.
• Xicohtencatl-Cortes J, Monteiro-Neto V, Saldaña Z, Ledesma MA, Puente JL, Girón JA. The Type 4 Pili of Enterohemorrhagic Escherichia coli O157:H7 Are Multipurpose Structures with Pathogenic Attributes. Journal of Bacteriology. 2009 Jan;191(1):411–21.
• Ueki T, Walker DJF, Woodard TL, Nevin KP, Nonnenmann SS, Lovley DR. An Escherichia coli chassis for production of electrically conductive protein nanowires. ACS Synthetic Biology. 2020 Mar 3;9(3):647–54.
• Sauvonnet N, Gounon P, Pugsley AP. PpdD Type IV Pilin of Escherichia coli K-12 Can Be Assembled into Pili in Pseudomonas aeruginosa. Journal of Bacteriology. 2000 Feb 1;182(3):848–54.
• Yousef H, Alhajj M, Sharma S. Anatomy, Skin (Integument), Epidermis [Internet]. PubMed. Treasure Island (FL): StatPearls Publishing; 2022. Available from: https://www.ncbi.nlm.nih.gov/books/NBK470464/
• Grenier D. Collagen-binding activity of Prevotella intermedia measured by a microtitre plate adherence assay. Microbiology. 1996 Jun 1;142(6):1537–41.
• Smith DR, Wang RS. Glutaraldehyde exposure and its occupational impact in the health care environment. Environmental Health and Preventive Medicine. 2006 Jan;11(1).
• Information Commissioner's Office (2019). The Data Protection Act. [online] Available at: https://ico.org.uk/media/2614158/ico-introduction-to-the-data-protection-bill.pdf