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Safety

Lab Practice

During this special edition of iGEM, we had access to the lab to perform our experiments. As safety was and is a major concern in science and for iGEM, we made sure proper training was received before accessing the laboratories. Following a lab safety course by a workplace safety officer, wherein we were instructed on how to handle and dispose biological waste, and to follow emergency protocol when deemed necessary. Further, training was given by our assistants to be able to work in a BSL-1 lab. Later in our project, we started working with mammalian cells in a BSL-2 facility and beforehand we were also taught those respective safety instructions by our assistant .

Some of the good laboratory practice and topics include:

Use of PPE such as lab coats, gloves, and safety glasses when needed.
Safe disposal of biological waste in BSL1/2
Safe handling of hazardous chemicals
How to act in case of health emergencies such as a wound, burning, or sensitive liquid in contact with the skin and eye.
Wearing a mask and maintaining recommended distance according to WHO, at work place during the COVID-19 pandemic

Safety of our design

From the start of our project, biosafety was a major focus of our project. As described in our design, we wanted to use a non-pathogenic carrier organism with the capability to colonize the gut, target and colonize cancerous tumours, and overall as low risk as possible for humans. Based on current literature, we found E.coli Nissle 1917 to be one of the best candidates. Indeed, being one of the first E.coli strains used as a probiotic [1], its effect and interaction with the gut microbiota have been well documented and thus risk of unwanted spreading within the human body is limited [2]. In addition, this particular strain has already been used for therapeutic delivery by scientists [3], [4], [5] as well as multiple iGEM teams over the years [6]–[8]. Finally, the innate ability of E. coli Nissle 1917 to colonize the tumour microbiome[9], [10] in addition to being a BSL-1 organism made it our most suited candidate. Finally, we specifically worked with a colibatine gene deleted E.coli Nissle 1917Δclb strain. In fact, Colibactin has been linked by scientists as a possible pro-carcinogenic compound [11].

In addition to using E.coli Nissle 1917, other E.coli strains such as E.coli NEB Turbo, E.coli NEB5α, E.coli DHL708 (Addgene #98417) were also used either for cloning or experimentation. All of these are BSL-1 organisms and not known to cause disease in healthy animals.
With the aim of proving the anticancer effect of our designed construct, we cultured several mammalian cancer cell lines: colorectal adenocarcinoma human epithelial cell lines Caco-2 (ATCC HTB-37). As this cell line requires aseptic handling to limit contamination.Therefore, we handled those in a BSL-2 lab under a laminar flow hood, even if they are classified as BSL-1.

We choose Azurin, as our antitumor compound,, a copper-binding redox protein isolated from P. aeruginosa that has been recently investigated as an inhibitor of p53. One of the strongest advantages of this protein is the absence of immunogenicity or toxicity in human cells [12], [13]. Additionally, we believe that by using chronotherapy, we can effectively reduce the emergence of resistance and maintain an acceptable level of efficiency compared to a continuous treatment[14]. Taking these assumptions into account, we aim to reduce the harmful potential of our construct.

Risk evaluated

Finally, during our risk assessment, we identified the following risks arising from our design:

Spreading inside and outside of the body: We aim to administer our therapeutic bacteria orally and it is known that a natural turnover occurs after colonization of the gut by the probiotic strain. Due to these effects, some of our designed constructs could get out of the body and thus spread to the environment. In addition, upon metastasis within the human body our bacteria may cause septic shock, an acute infection in blood .
Horizontal gene transfer

Our engineered bacteria are aimed to reside inside the tumoral microbiome, where it is likely to encounter other microorganisms. As a consequence, we cannot exclude the risk of horizontal gene transfer between bacteria. Hence, one of our main concerns is the spreading of antibiotic resistance genes, used during the cloning phase of our project and the transfer of the other genes involved in our design.

What We Did to Counter Them

In an effort of containing the evaluated risks, we designed a versatile kill switch. By using a chemical and temperature inducible gene circuit, we are able to prevent the spread of our engineered bacteria. In addition, using a toxin/antitoxin system allows us to reduce the use of antibiotic resistance as a selection tool during cloning. Learn more about it on our Kill switch page .

References (click to see)

J. Behnsen, E. Deriu, M. Sassone-Corsi, et M. Raffatellu, «Probiotics: Properties, Examples, and Specific Applications», Cold Spring Harb. Perspect. Med., vol. 3, n^o 3, p. a010074, janv. 2013, doi: 10.1101/cshperspect.a010074.
U. Sonnenborn et J. Schulze, «The non-pathogenic Escherichia coli strain Nissle 1917 – features of a versatile probiotic», Microb. Ecol. Health Dis., vol. 21, n^o 3‑4, p. 122‑158, janv. 2009, doi: 10.3109/08910600903444267.
P. J. Sarate et al., «E. coli Nissle 1917 is a safe mucosal delivery vector for a birch-grass pollen chimera to prevent allergic poly-sensitization», Mucosal Immunol., vol. 12, n^o 1, Art. n^o 1, janv. 2019, doi: 10.1038/s41385-018-0084-6.
C. Pöhlmann et al., «Improving health from the inside», Bioengineered, vol. 4, n^o 3, p. 172‑179, mai 2013, doi: 10.4161/bioe.22646.
P. Praveschotinunt, A. M. Duraj-Thatte, I. Gelfat, F. Bahl, D. B. Chou, et N. S. Joshi, «Engineered E. coli Nissle 1917 for the delivery of matrix-tethered therapeutic domains to the gut», Nat. Commun., vol. 10, n^o 1, Art. n^o 1, déc. 2019, doi: 10.1038/s41467-019-13336-6.
«Team:Penn/Nissle - 2012.igem.org». https://2012.igem.org/Team:Penn/Nissle (consulté le oct. 20, 2020).
«Chassis: E. coli Nissle 1917». https://2013.igem.org/Team:Arizona_State/Nissle (consulté le oct. 20, 2020).
«Team:TAS Taipei/Applied Design - 2018.igem.org». https://2018.igem.org/Team:TAS_Taipei/Applied_Design (consulté le oct. 20, 2020).
J. Stritzker, S. Weibel, P. J. Hill, T. A. Oelschlaeger, W. Goebel, et A. A. Szalay, «Tumor-specific colonization, tissue distribution, and gene induction by probiotic Escherichia coli Nissle 1917 in live mice», Int. J. Med. Microbiol., vol. 297, n^o 3, p. 151‑162, juin 2007, doi: 10.1016/j.ijmm.2007.01.008.
Y. Zhang et al., «Escherichia coli Nissle 1917 Targets and Restrains Mouse B16 Melanoma and 4T1 Breast Tumors through Expression of Azurin Protein», Appl. Environ. Microbiol., vol. 78, n^o 21, p. 7603‑7610, nov. 2012, doi: 10.1128/AEM.01390-12.
L. Jia et al., «Preclinical pharmacokinetics, metabolism, and toxicity of azurin-p28 (NSC745104) a peptide inhibitor of p53 ubiquitination», Cancer Chemother. Pharmacol., vol. 68, n^o 2, p. 513‑524, août 2011, doi: 10.1007/s00280-010-1518-3.
N. Bernardes, A. S. Ribeiro, R. Seruca, J. Paredes, et A. M. Fialho, «Bacterial protein azurin as a new candidate drug to treat untreatable breast cancers», in 1st Portuguese Biomedical Engineering Meeting, mars 2011, p. 1‑4, doi: 10.1109/ENBENG.2011.6026047.
D. Adam, «Core Concept: Emerging science of chronotherapy offers big opportunities to optimize drug delivery», Proc. Natl. Acad. Sci., vol. 116, n^o 44, p. 21957‑21959, oct. 2019, doi: 10.1073/pnas.1916118116.

A big thank you to our sponsors for their valuable support!