Safety

General Safety

Our laboratory work is conducted at Lab 106 of the National Taiwan University’s Synthetic Biology and Biofabrication Laboratory, a Biosafety Level 1 lab operated by Professor Hsuan-Chen Wu. We were supervised mainly by students of National Taiwan University. Because the lab is of Biosafety Level 1, it only permits the use of agents that don’t pose significant health risks upon human exposure. Thus, we only worked with common bacterial strains DH5ɑ and BL21(DE3) throughout our experiment. Our experiment was conducted under constant supervision by graduate students and trained lab technicians.

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Before performing labwork, we made experiment plans that were reviewed and validated by our instructors to prevent unnecessary risks during experimentation. All plans are uploaded onto a shared drive to keep instructors and team members informed of the experiment details and schedules. We made certain that all general lab-safety guidelines of Lab 106 were followed and all equipment was used with caution. Experiments with microorganisms were conducted in laminar flow hoods, and the glassware used was disinfected with diluted bleach and rubbing alcohol upon experiment completion. In the case of bacterial contact with our workspace, rubbing alcohol was immediately applied to any contaminated areas and wiped clean. Chemical waste was disposed of in biohazard waste bins; chemicals, pipette tips, and glassware were sterilized in the autoclave.

Project Specific Safety

Bacterial Strain

The bacterial strains we used in actual experimentation are commercial DH5ɑ and self-prepared BL21(DE3) competent cells. The former is for plasmid amplification and the latter is for protein expression. We further planned to transfer the assembled plasmids to B. subtilis - our ideal chassis - but no such experiments were actually conducted. The bacteria we manipulated pose little harm to adult humans even in the case of environmental contamination and skin contact, as they are of Risk Group 1. Nevertheless, we still closely followed general lab-safety procedures to minimize danger to ourselves. Upon completion of any experiments directly involving the use of bacterial strains, all waste is disposed of in biohazard waste bins.

Pathogen

Though we originally planned to experiment with FOC, we ultimately decided against those plans due to the potential hazard of environmental contamination. Due to the sturdiness of the FOC spores, effective damage control as FOC contacts the soil would be extremely unlikely. Thus, we replaced our co-culture antagonism assay with yeast (Saccharomyces cerevisiae) to test the antifungal capabilities of our engineered bacteria due to its lower safety risk.

Public Acceptance and Safety

From Figure 1., our survey showed that 67.1% out of the 1500+ surveyed individuals harbor some sense of distrust towards genetically modified fruit. Feedback from our in-person interviews also showed significant skepticism towards genetically altered plants, with many preferring organic fruits. We decided to engineer probiotic bacteria external to the banana plant to stop Fusarium Wilt after seeing that genetic alteration to the banana itself is optically unfavorable.

Product Specific Safety

Regulations

Given that our probiotics will be applied directly to the soil, we need to check whether the regulations enable the use of our probiotics directly on the banana root and in the field. Because any organism that either directly or indirectly harms plants is referred to as a pest and hence banned from field or plant application, we have to prove that our probiotic can't harm organisms other than Foc TR4 in a significant manner. Since our theorized chassis B. subtilis is a common, benign soil microbe, it is essentially considered to be nonpathogenic (Olekar et at., 2012), so this concern is reduced; however, the genetic engineering involved in our bacteria makes it such that it still needs to pass numerous strict regulations outlined in the Plant Protection and Quarantine Act (Council of Agriculture, 2018). Extensive tests will be required to prove the effectiveness and harmlessness of our probiotic.

An alternative method of implementation could be considered to bypass these safety concerns. While genetically engineered bacteria have to undergo extensive safety testing to reach approval for implementation, the recombinant proteins produced do not face the same level of restrictions. Hence, an alternative implementation method exclusively applying recombinant Chi18h8 may help us avoid gridlock on the regulation front.

Product Ingredient Safety

The safety of our primary ingredient, genetically engineered B. subtilis, is discussed in the section above. Though most of the other ingredients in our theorized product are essentially fertilizer, harm could still be caused to plants if the quantity applied is excessive.(Weisenhorn & Hoidal, 2021). Hence, we kept the concentration of all nutrients at 4%-5%; this was a decision made by referencing similar products (Roots inoculant, Dekker, 2016; Worm Tea, Microbe Brew). We avoided the inclusion of animal-derived protein hydrolysates in the ingredient list due to potential phytotoxic effects and growth depression and opted instead for plant-derived counterparts. The inclusion of kelp extract for fertilization is proven to cause no harm to plants. (Flower, 2021).

References