Safety

Safety first! Since we wholeheartedly support this statement we have constantly dealt with all kind of safety issues arising during our project. Our aim was to develop an autonomous and sustainable mosquito trap called S.H.I.E.L.D. Therefore, we equipped E. coli bacteria with resistance plasmids containing the genetic information for odour baits, growth inhibition and an insecticide. Working with genetically modified organisms (GMOs) implied the constant verification of the safety of people and environment. As we would like to establish the S.H.I.E.L.D. in the field, we had to take special care to ensure GMOs are not released into the environment in case of damage to the trap. However, in the case of a release despite our efforts the measures described in the following article will prevent them from proliferating uncontrolled. Furthermore, we would like to show general safety measures we took while working in the lab as well as the considerable thoughts we put into the safe design of the S.H.I.E.L.D.

Safe Lab Work

Most important for conducting experiments safely, is an appropriate planning of experiments in advance. Therefore, we had weekly lab meetings, during which we planned the experiments of the following week.

Regarding our working space, we are very happy, that we were provided with an amazing biochemistry lab from the working group of Prof. Zoya Ignatova. All of our cloning work and characterizations were carried out in this room (Fig. 1) at safety level one. Before we started working in the laboratory, we conducted a safety training including emergency contacts and routes.

**Fig. 1 Biochemistry lab.** Our own workbench provided to us by the working group of Prof. Zoya Ignatova where we spend the last half year. It fulfils all safety conditions.

During our lab work, we always wore appropriate protective clothing such as lab coats and safety eyewear. We were aware, that a lot of substances used to stain DNA or proteins are potentially cancerous and therefore special precautions are needed while working with them. Staining of DNA with ethidium bromide was carried out only in separate, contaminated areas where we always wore certified gloves (Fig. 3).

**Fig. 3: Cutting out bands.** After restriction we isolated the DNA applying the gel purification after gel electrophoresis. For this we used gloves and eye protection to protect ourselves from the UV light and ethidium bromide.

The work on the hydrogel was conducted in a specialized chemistry lab at the faculty of pharmacy. Before working in this lab we were instructed regarding additional safety precautions. Of course the synthesis was performed under a standard fume cupboard to minimize potential harm from the chemicals used. We were aware of using potentially cancerous and poisonous substances (e. g. the monomers 4,4’-oxydianiline (ODA)) and paid particular attention to them. Flammable or hazardous substances were stored in according safety cabinets.

While conducting experiments on mosquitoes, we were allowed to use a laboratory situated at the Bernhard Nocht Institute for Tropical Medicine. Since we did not work with disease-transmitting animals a safety level one laboratory was sufficient.

Safe Project Design

Hardware

Besides general safety measures during our work in the lab we had to consider many issues specific to our project. Especially due to its planned application in the field we integrated several safety features into our hardware. To prevent breakage and leaks under strong environmental influences, and forces we plan on using a plastic that is very hard, durable, UV resistant, autoclavable as well as strong sealing rings.

To make sure the GMOs stay contained within the S.H.I.E.L.D. we build a nanofilter between the two hydrogels. The filters pore size allows the passage of small substances such as the odor baits and the insecticide while preventing bacteria and microorganisms to exit or enter the trap. This also means that there is no possibility of mosquitoes sucking GMOs out of the trap.

In addition, the hydrogel is antimicrobial whereby a growth of funghis is excluded. A fungal film would inhibit the diffusion of the substances and could discourage the mosquitoes from stinging into the gel. Furthermore, the antimicrobial nature of the hydrogel ensures that pathogens can not be distributed by mosquitoes landing on the gel. Dr. Jacobs, mosquito expert from the Bernhard Nocht Institute for Tropical Medicine, assured us the growth of pathogens on our gel is highly unlikely.

As moisture improves the hydrogels durability, we made sure to design the upper part of the S.H.I.E.L.D. in a way that enables condensation water to be conducted to the hydrogel. An intact hydrogel is not only important for the S.H.I.E.L.D.’s functionality but it also creates a further layer of protection between the GMOs and and the environment. The self-healing properties of the gel ensure its integrity even after mosquitoes sting into it.

Additionally, the gel also guarantees that only animals that can sting the hydrogel and suck the toxin out are impacted by the S.H.I.E.L.D. The specificity of our mosquito trap is further enhanced by the lures used. Further information can be found in the section Risk assessment of our bacteria.

Risk assessment of our bacteria

We used DH5α, a derivative of E. coli wild type strain belonging to risk group 1 and thus classified as low risk. To help us with our further risk assessment we invited Dr. Mirko Himmel from the Carl Friedrich von Weizäcker-Centre for Science and Peace Research. He queried how we would prevent the uncontrolled spread of our bacteria should leakage occur in spite of the considerable efforts we made to prevent this in our hardware design. Growth inhibition of the GMOs not only enables the sustainability of the S.H.I.E.L.D. but also prevents the proliferation of the GMOs in case of leaks in the trap. Our growth inhibition module BBa_K2588021 inhibits DNA replication, peptidoglycan synthesis, formation of FTSZ rings, and cell elongation. While the different methods of growth inhibition would be lethal by themselves, by introducing all of them combined into the cell we stabilized the cell culture. This further ensures that the GMOs would not be able to grow if a mutation within the growth module should occur. While variation in only one of the parts might still be stable, mutations in multiple of them would be lethal to the cell.

Dr. Himmel also suggested to rethink the insecticide we originally chose to implement. The data for the first toxin suggested a minimal toxicity to mice. Since we did not have enough information on either the concentration of the toxin needed to be harmful to mammals nor the expected maximal concentration in the S.H.I.E.L.D. or the lab we decided to use a different insecticide. BjalT, the insecticide we chose, is a neurotoxin that is derived from the Judean Black Scorpion. It acts on voltage-gated sodium channels of insects only and is therefore not harmful to mammals. Thus, it meets the criteria to work with it in a security level one lab. Even though the toxin has a rather long half-life due to its small size and multiple disulphide bonds we preclude any significant impact on the environment by the toxin. The amounts ingested by the mosquitoes that is released into the environment after their death would be present only in small doses and would further decrease with ascending levels of the food chain. The specificity of the toxin to insects is a further safeguard.

Additionally, the composition of the lure is designed to mimic the odours emanating from the human skin. Thus mainly human-biting animals should be lured into the S.H.I.E.L.D.

To verify the effectiveness of our lure, the toxin and the hydrogel on mosquitoes we are working at the Bernhard Nocht Institute for Tropical Medicine, where the laboratories are especially equipped for experiments like ours. Furthermore we are working with Anopheles gambiae that have not been infected with plasmodium and are therefore harmless to humans.

During the interview with Dr. Himmel we talked about the possibility of the S.H.I.E.L.D being misused with malicious intent. However, to use the trap as a bioweapon extensive expertise would be necessary. Not only would the hardware design need to be adjusted but the E. coli would have to be altered. The toxin would have to be replaced by another one and the growth inhibition module would have to be deactivated since the S.H.I.E.L.D is not designed to produce great amounts of toxin.

Future Steps

Before testing the S.H.I.E.L.D. in the field we would introduce further safety measures.

Currently we are working with antibiotics staggered medium to ensure the plasmids are not ejected by the GMOs. However, the S.H.I.E.L.D. cannot rely on antibiotics when establishing it in the field. Thus, we are planning to integrate the DNA into the bacterial genome, relinquishing the need for antibiotics.

To ensure that GMOs cannot spread in case of the S.H.I.E.L.D.’s destruction we would introduce a kill switch such as the Amber strain of Team Darmstadt 2016.

A stop codon (Amber codon) ist introduced in a transcription factor responsible for inhibiting a lethal gene. An amino acid that corresponds with the Amber codon and does not usually occur in E. coli would be produced by the cyanobacteria. Should the trap break and the two cultures would no longer be in cohabitation the amino acid would no longer be provided to E. coli. Thus, the lethal gene would be expressed and E. coli would die. Even in the case of a simultaneous release, the cyanobacteria would not be able to survive under African climatic conditions, and thereby both cultures would die.

One of the issues we still have to find a suitable solution for is the transport of the S.H.I.E.L.D from the place of production to the site of application. It should be handled with care and due to the Amber strain the cell cultures have to be transported already in a working symbiosis.

Since we were approached multiple times with the question of whether we were planning to exterminate the mosquitoes we would like to take this opportunity to answer it with a clear no. Our goal is to employ the push and pull principle to create a local malaria free zone thus protecting the people and controlling the transmission of malaria.

After implementing all these aspects in the S.H.I.E.L.D. it will be ready to safely combat malaria.