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Safety

The general public has many misconceptions when it comes to genetically modified organisms (GMOs), which then leads to a lot of fear and stigma. In terms of gaining widespread acceptance for synthetic biology applications in daily life, there is still much effort to be made. Since the advent of synthetic biology, scientists have been working hard to implement safeguards in their genetically modified organisms. When it comes to biotherapeutics or any product of genetic engineering that will be consumed by humans, safety has never been more important.

Laboratory Safety

The iGEM NCKU-Tainan team is in full compliance with the safety and security rules of the iGEM competition.

The team was included in the laboratories of Prof. Masa Hashimoto and Prof. I-Hsiu Huang, which contains equipment for both Biosafety Level 1 and 2. Before we started our work in the lab, all team members had to receive training and pass different safety tests provided by our university. We also received training from our advisors on how to use the machinery in the lab (i.e. thermocycler, electroporation machine) and also on the use of chemicals (i.e. p-Cresol, staining reagents). At all times, a supervisor or instructor was present when work was conducted in the laboratory.

Project Safety

Escherichia coli Nissle 1917

In order to make our biotherapeutic safe for human consumption, we used E. coli Nissle 1917 as our chassis. Escherichia coli Nissle 1917 is a nonpathogenic E. coli strain isolated by Alfred Nissle in 1917. It is one of the best examined probiotic strains, used in many gastrointestinal disorders including diarrhea, uncomplicated diverticular disease and ulcerative colitis (UC).^[1] It has proven to have an intestinal anti-inflammatory effect without major immunotoxic properties.^[2,3] A clinical study to evaluate the clearance of E. coli Nissle has proven that all subjects cleared E. coli Nissle with a median clearance of 1 week making it beneficial for therapeutic probiotic because it is not expected to colonize the gut for a long time.^[4]The safety, tolerability, and efficacy of consuming E. coli Nissle have also been evaluated and research has shown that daily treatment with E. coli Nissle for chronic disorders is feasible.

Kill Switch

In order to make our live therapeutic safe for human consumption, we added a kill switch into our E. coli Nissle in the form of gene knockouts. We used Lambda Red Recombineering system, which is based on homologous recombination, to perform gene knockouts. By using lambda red genes and hijacking E. coli Nissle’s own recombineering system, we are able to replace the target DNA region with a DNA fragment of our choosing. Electroporation was then used to deliver DNA fragments and plasmids into the bacteria (link to protocols).

We amplified the Chloramphenicol Resistance (CmR) Cassette from pKD3 plasmid using Amplification PCR, and used that fragment to replace the target gene. The resistance cassette is flanked by FRT sites, which then allows the removal of the cassettes with the help of pCP20, an FLP helper plasmid. Before we electroporated the CmR cassette into E. coli Nissle, we electroporated in pKD46, a recombinase helper plasmid.

We experimented with two different gene knockouts, can gene knockout and dapA gene knockout, and compared its effectiveness.

dapA Gene Knock Out

dapA encodes for 4-hydroxy-tetrahydrodipicolinate synthase, an enzyme necessary for cell wall synthesis.^[5] By knocking out this gene, the bacteria will have to depend on exogenous diaminopimelate (DAP) for cell wall assembly and growth, making it a potential kill switch as the bacteria will not survive without suppliance of exogenous DAP.

We confirmed that we have successfully replaced the dapA gene with CmR cassette using colony PCR. The size of CmR cassette inserted between the homology arms is approximately 1600 base pairs.

Fig. 1. (a) Schematics showing dapA knockout strategy (b) Confirmation of dapA knockout in E. coli Nissle via colony PCR. M: Marker; Lane 1: Wild Type (no bands); Lane 2: ΔdapA::CmR 1 (1.6 kb); Lane 2: ΔdapA::CmR 2 (1.6 kb)

To further confirm our mutant E. coli works as a kill switch, we did a phenotype test by streaking out bacteria on different plates.

Fig. 2. Confirmation of dapA knockout in E. coli Nissle. E. coli Nissle and other strains were streaked onto agar plates containing (A) just Lysogeny Broth (LB); (B) 0.1 M DAP; (C) Chloramphenicol (Cm) and 0.1 M DAP for phenotyping.

As seen in Fig. 2, without exogenous DAP, ΔdapA::CmR is unable to grow. And since it can survive on Cm plates, we can confirm that it contains the CmR cassette.

However, we realized that we would have to think of a way to provide exogenous DAP in our capsule to prevent our E. coli from dying before it reaches the gut. After deliberating the cost-performance ratio, we decided it wasn’t a good fit for what we had in mind. Thus, we did not continue on with replacing the CmR cassette with FRT sites.

can Gene Knock Out

The can gene of E. coli encodes for carbonic anhydrase (CA), an enzyme that assists rapid interconversion of CO₂ and water into carbonic acid, protons and bicarbonate ions. E. coli requires a constant supply of bicarbonate as a metabolic substrate during normal growth.^[6,7]So, if this gene is knocked out, E. coli is unable to turn the CO₂ into bicarbonate fast enough before the CO₂ diffuses out and causes cell death. Inside the gut, where we want the E. coli to survive, the CO₂ concentration is high enough (ranging from 5% to 29%) to allow the spontaneous conversion of CO₂ into bicarbonate ions. However, when E. coli exits the human body, the lowered concentration of CO₂ will result in its death.

We confirmed that we have successfully replaced the can gene with CmR cassette using colony PCR. The size of CmR cassette inserted between the homology arms is approximately 1400 base pairs. The size of FRT sites inserted between the homology arms is approximately 500 base pairs.

Fig. 3. (a) Schematics showing can knockout strategy (b) Confirmation of can knockout in E. coli Nissle via colony PCR. M: Marker; Lane 1: Wild Type (~1.1 kb), Lane 2: Δcan::CmR 1 (~1.4 kb), Lane 3: Δcan::CmR 2 (~1.4 kb), Lane 4: Δcan::FRT 1 (~500 bp), Lane 5: Δcan::FRT 2 (~500 bp)

To further confirm our mutant E. coli works as a kill switch, we did a phenotype test by streaking our bacteria on different plates and placing them in different conditions.

Fig. 4. Confirmation of *can* knockout in *E. coli* Nissle. *E. coli* Nissle and other strains were streaked onto agar plates and placed in (A) 0.04% CO₂; (B) 5% CO₂; (C) 5% CO₂ and contains Chloramphenicol (Cm) for phenotyping.

As shown in Fig. 4, Δcan::CmR and Δcan::FRT requires a higher CO₂ level to survive. When streaked on Cm plates, Δcan::CmR is still able to grow while Δcan::FRT is unable to grow, thus proving that we have succeeded in knocking out the can gene and replacing it with CmR cassette and FRT sites respectively.

Thereby, we will use the can gene knockout as our kill switch.

Device Safety

This year, iGEM NCKU Tainan designed CreSense, a user-friendly, affordable and customizable biosensing device, that has the potential to be placed in diagnostic centres or other healthcare service providers. However, as a medical device, safety and effectiveness plays a critical role in healthcare because defective or unsafe medical devices may cause severe consequences to happen. Because our device contains engineered bacteria, we have taken adequate precautionary measures to ensure CreSense will not cause hazards to both users and the environment when people use them.

Bio-Sensing bacteria

Bio-Sensing bacteria is one of the major key players in CreSense. To ensure its viability for long-time storage and transport purposes, the bacteria will be lyophilized by suspending log-phase cells in a lyophilization medium and then freeze-drying the suspension. All the users have to do is to refresh the bacteria pallet and inject the culture into the microfluidic chip.

Microfluidic disc

Our microfluidic disc is designed for one-time use to avoid decontamination or sterilization problems. The disc is also designed in a way that allows the bacteria to be injected into the disc, but not allow the bacteria to flow out. Our microfluidic disc will then be disposed of as biomedical waste after being used, as it not only contains engineered bacteria but also blood.

References

Sonnenborn, U., & Schulze, J. (2009). The non-pathogenic Escherichia coli strain Nissle 1917 – features of a versatile probiotic. Microbial Ecology in Health & Disease, 21(3-4).
Kurtz, C. B., Millet, Y. A., Puurunen, M. K., Perreault, M., Charbonneau, M. R., Isabella, V. M., … Miller, P. F. (2019). An engineered E. coli Nissle improves hyperammonemia and survival in mice and shows dose-dependent exposure in healthy humans. Science Translational Medicine, 11(475).
Scaldaferri, F., Gerardi, V., Mangiola, F., Lopetuso, L. R., Pizzoferrato, M., Petito, V., … Gasbarrini, A. (2016). Role and mechanisms of action of Escherichia coli Nissle 1917 in the maintenance of remission in ulcerative colitis patients: An update. World Journal of Gastroenterology, 22(24), 5505.
Kurtz, C., Denney, W. S., Blankstein, L., Guilmain, S. E., Machinani, S., Kotula, J., … Brennan, A. M. (2017). Translational Development of Microbiome-Based Therapeutics: Kinetics of E. coli Nissle and Engineered Strains in Humans and Nonhuman Primates. Clinical and Translational Science, 11(2), 200–207.
InterPro EMBL-EBI. “4-Hydroxy-Tetrahydrodipicolinate Synthase, DapA (IPR005263) < InterPro < EMBL-EBI.” Ebi.Ac.Uk, 2019, www.ebi.ac.uk/interpro/entry/IPR005263. Accessed 5 July 2019.
Merlin, C., Masters, M., McAteer, S., & Coulson, A. (2003). Why Is Carbonic Anhydrase Essential to Escherichia coli? Journal of Bacteriology, 185(21), 6415–6424.
Hashimoto, M., & Kato, J.-I. (2003). Indispensability of the Escherichia coli Carbonic Anhydrases YadF and CynT in Cell Proliferation at a Low CO₂ Partial Pressure. Bioscience, Biotechnology, and Biochemistry, 67(4), 919–922.

Team:NCKU Tainan/Safety