Lab Safety

When working and implementing GMOs, safety is the number one priority. It is imperative that all regulations are strictly followed to prevent any unwanted release of the GMO into the environment. With recommendations and assistance from our supervisor and staff at our laboratory, we maintained a safe level of conduct during our work with GMO bacteria, thereby preventing any contamination.
Our labs enabled us to work in a minimum containment level (ML) 1 lab. To minimize contamination risks, only two lab members worked in this lab at any given time, changing protective clothing and gear before entering and leaving the ML-1 lab. All the laboratory team members received brief safety training by our advisor.

The training included, but was not limited to:

Proper handling of the laboratory equipment and devices.
The necessary usage of personal protective equipment (PPE).
Taking the steps needed to prevent fire hazards, and how to deal with a fire in the event of one.
Strict hygiene norms: no food or drinks of any kind in the laboratory, amongst others.
Correct disposal of any hazardous materials.
The protocol to follow in case of spillage of and/or human exposure to hazardous materials.

The use of headphones or other devices that could hinder communication were prohibited. Before entering or leaving the laboratory the sterilization steps according to the lab guidelines were followed to prevent the spread of microorganisms into the environment. To avoid cross-contamination the laboratory surfaces were disinfected frequently.

Project Design

General Microorganism Information:

All the microorganisms that were used in this project were classified to risk group 1 and were always handled with the corresponding protocols. These include well-characterized and non-pathogenic Escherichia coli strains (E.coli BL21 and DH5ɑ).

Specific Project Safety:

Kill switches were implemented to ensure the safety of our GMO. The necessary sequences for the specific genes intended to use were researched. Using SnapGene a hairpin structure was developed which is associated with a nucA (BBa_k11159105), which degrades all the dsDNA that is present in the organism. Another measure created using SnapGene was the gate, in which a H2O2 sensitive promoter would regulate the transcription of bromoform producing proteins.

In vitro

After extensive testing in the lab, environmental testing is planned as the next step, which would allow for the testing of our biosafety measures. Yet an initial in vitro simulation will first be used, as this is a less radical experimentation approach. The ‘Gut in Glass’ simulation, will be tested in the ML-1 lab to analyze the levels of bromoform produced by our bacteria, the H2O2 levels and CH4 as well as the efficacy of the kill switches developed. An initial testing of the rumen simulation concept can be found under Awards: Best Model.

Once the mentioned procedures have occurred and adjustments are made to generate positive results, animal testing may be initiated. This will be extensively regulated and the rules of the country of testing will be followed. This research would be conducted in the Netherlands therefore the team will be in contact with the Dutch Ministry of Public Health and the Environment (RIVM), and the animal ethics research committee of our university.

In vivo: Closed Stage

Adult dairy cows will be used to test our feed additive. The cows must be dairy cows to allow for testing of bromoform contents in the milk. To protect the soil and the surrounding areas the cows will be placed in an inside room fitted with a breathing chamber as used in Alemu et al. (2017) to measure methane emissions. This inside space will inhibit the spread of bacterial cells through the soil. The floor and walls will be fitted with a membrane, which can be burned at the end of the experiment.

Storage, transportation, and rules for the use of GMO products by animals - all these delivery stages require compliance with safety rules.
The final type of MethaGone as a food additive has the form of a gelatin capsule with a mixture of bran, molasses, and bacterial cultures inside. Gelatin capsules are widely used to implement powdered food additives into cattle diets, and the composition of the powder inside ensures the viability of bacteria inside for up to 45 days (Supratman et al., 2020).
Consequently, each cow will be provided with a dose of the supplement through their feed. The capsules prevent the release of the bacteria in the gastric tract prior to the rumen. Storage of such capsules and many bacterial cultures implies a cool dark place and similar conditions during transportation.

As described in the previous section, all measures must also be taken for the safe disposal of cow waste products, at least at the stage of establishing the safety of the product in order to obtain a license.

Specialized sanitation airlock entrances are available to allow researchers to enter and take milk, faecal, urine and saliva samples. If the cows have a perforated rumen, rumen fluid samples are also tested for presence of our bacteria, as well as the unwished presence of our plasmid in different bacterial species. Furthermore, non-treated cows can be added to the testing areas, and the same samples are taken from them. These tests allow for assessing potential cross-contamination between animals. In further tests, the surrounding areas are tested for presence of the bacteria in question.

The milk, and later also the meat, will be analysed for traces of bromoform, to ensure the safety of consumption. Necropsies will be carried out to test for abnormal, cancerous lesions or infected areas along the mucosal lining of the rumen, as well as the other intestines that are potentially in contact with the bacteria. Furthermore, animal weight gain, average dry matter intake, and faecal composition will be assessed to ensure the health of the animals throughout the experiment.

Alemu, A. W., Vyas, D., Manafiazar, G., Basarab, J. A., & Beauchemin, K. A. (2017).
Enteric methane emissions from low– and high–residual feed intake beef heifers
measured using GreenFeed and respiration chamber techniques1,2.
Journal of Animal Science, 95(8), 3727–3737. doi:10.2527/jas.2017.1501

Supratman, H., Ramdani, D., Joni, I. M., & Ismiraj, M. R. (2020).
Preparation and characterization of probiotics in powder form.
APPLIED PHYSICS (ICC-2019). Published.

In vivo: Semi-closed Stage

According to governmental legislation, more biosafety aspects can be included, and constant revision and experimentation will ensure a high safety standard before the next testing phase, which would be in a semi-open environment. In these experiments, cows would still be at a research facility, yet one that has an optional outdoor area for the animals. In some distance, the same safety appliances are still implemented, just as in the closed-environment experiment. Membranes underneath the soil prevent the bacteria from spreading, and net-structures surrounding the outside area prevent animal incursion. The same tests as in the previous experiments will be conducted, yet additionally, soil samples will be taken to ensure no trace of our bacteria or of our plasmid can be found.

Through long and thorough experiments and adaptations after assessing the results, we strive to achieve maximum biosafety, while still reducing methane emissions drastically, all while keeping the animals healthy.