Safety

Working in a safe environment - laboratory, GMO and cadmium

Working within a laboratory, particularly when dealing with genetically modified organisms (GMOs), necessitates stringent safety protocols. This was particularely important given that we were working with cadmium, a dangerous heavy metal. As such, we incorporated extra precautions throughout our experimental, waste disposal, and storage processes.

Prior to initiating our work with cadmium, our team undertook specialized laboratory training to ensure proficiency in both fundamental procedures and safety standards.

We consulted with the Occupational Safety and Health (OSH) department at EPFL to delineate specific safety protocols. We implemented distinct waste management practices for handling cadmium and carried out all experiments within a controlled environment, wearing essential protective equipments including a fume hood, P3 masks, lab coats, and safety goggles.

During our initial experimental phase, we opted for zinc as a proxy for preliminary assessments, given its chemically analogous properties to cadmium and its affinity for binding with EC20. This approach enabled us to calibrate various experimental parameters, including incubation duration, bacterial load, and temperature, thereby establishing optimal conditions. Furthermore, it facilitated our proficiency with spectrometric methodologies, while concurrently minimizing exposure to hazardous substances. In subsequent phases, we expanded our investigations to incorporate cadmium, leveraging insights derived from our earlier zinc-based experiments to ensure methodological efficiency.

We carefully selected our colorimetric test for safety considerations. The supplier's cuvettes are specifically engineered to mitigate exposure risks, and the responsibility of waste management resides with the provider.

We have documented all our safety measures for working with cadmium in the following document, for subsequent iGEM teams. Unfortunately, the Incidin Pro document is in French, because the company did not provided any infomative document in English.

Avoiding modified bacteria propagation

Auxotrophy

In order to achieve its intended effects, our genetically modified bacteria will come into contact with various aspects of the human body, including the digestive tract and the microbiota. Given the genetic modification, it is imperative to ensure that these bacteria do not disrupt bodily functions, colonize the microbiota, or persist in the external environment.

To address this concern, we initially conducted all proof of concept experiments using the laboratory strain of E. coli. However, the ultimate goal is to employ human intestinal E. coli strains orLactobacillus for the final product. These strains are naturally present in the intestinal tract, which mitigates the risk of adverse reactions from the body and unwanted effects.

Furthermore, we plan to utilize auxotrophic strains. In the intestinal medium, there are amino acids present, but their concentration is insufficient to support bacterial growth. This strategic choice ensures that the bacteria can survive their journey through the body but will be incapable of growth, division, and proper colonization. This approach effectively prevents microbiome colonization and disturbance. Moreover, as the bacteria exit the body, their inability to access adequate amino acids will ultimately lead to death.

Mucus-binding management

As explained on the engineering page, we have developed a system that prevents the production of SpaC outside the laboratory medium. This control is achieved through the use of IPTG and theophylline. After the freeze-drying process of the probiotic, SpaC production is halted as the metabolism of the bacteria stop but protein would be preserved. Since the half-life of SpaC falls within the range of approximately 48 hours, the bacteria will have a limited window of time during which they can adhere to the mucus after reactivation through hydration. In addition, human mucus undergoes a natural turnover process, which serves as an additional mechanism for expelling bacteria from the body, even if they are attached to the mucus.

To go further...

This ensures that all genetically modified bacteria are expelled from the body and ultimately perish. Our final concern revolves around horizontal gene transfer (HGT). Indeed, even if our genetically modified bacteria perish, the possibility of gene transfer between microbiotal strains and our bacteria before their demise remains a concern. Such transfer, involving one of our plasmids, could enable other bacteria to bind cadmium, produce SpaC, and develop antibiotic resistance. In the case of SpaC, the presence of IPTG and theophylline would still be required, making this occurrence less likely. Regarding EC20, it not anticipated to confer a particular advantage to the bacteria. However, we utilize two different antibiotic in our plasmids : ampicillin kanamycin. The primary issue is the development of antibiotic resistance.

Avoiding horizontal gene transfer : Dut/Ung mutant

The importance of LBP for the upcoming years

Many diseases result from intricate interactions between metabolic and immunological processes, presenting significant medical challenges. There is a growing recognition of the role of microbes in human health and disease. Evidence is accumulating that engineered microbes, a new class of live biotherapeutics, could target disease mechanisms effectively. Leveraging synthetic biology, nonpathogenic bacteria can be engineered to detect environmental signals, eliminate harmful substances, and deliver therapeutic agents. In this perspective, we discuss key factors to consider in the design and development of engineered live biotherapeutics to gain regulatory approval and patient acceptance.¹

Present dangers with bacteria

Over the past decade, antibiotic resistance genes (ARGs) have significantly heightened the microbial threats to human health. The evaluation of the relative risks associated with these ARGs is a complex task, as numerous genes can impart resistance. Factors such as the prevalence of ARGs, their potential for lateral transfer, and their ability to be expressed in pathogenic microorganisms all contribute to their importance. In some studies, they identified a total of 2,561 ARGs that collectively conferred resistance to 24 different classes of antibiotics. Approximately 23.78% of the identified ARGs indeed present a health risk, particularly those responsible for multidrug resistance. ARGs are a threat to both human and animal health.²

Our final concern revolves around horizontal gene transfer (HGT). Indeed, even if our genetically modified bacteria perish, the possibility of gene transfer between microbiotal strains and our bacteria before their demise remains a concern. Such transfer, involving one of our plasmids, could enable other bacteria to bind cadmium, produce SpaC, and develop antibiotic resistance. In the case of SpaC, the presence of IPTG and theophylline would still be required, making this occurrence less likely. Regarding EC20, it not anticipated to confer a particular advantage to the bacteria. However, we utilize two different antibiotic in our plasmids : ampicillin kanamycin. The primary issue is the development of antibiotic resistance.

Uplasmid : a promising tool for LBP

To prevent HGT, we developped the Dut/Ung system.

In bacteria such as Escherichia coli, dUTP is involved in the pathway for de novo synthesis of dTTP. Since DNA polymerases is not perfect, it makes mistakes and uses dUTP in place of dTTP, some incorporation of dUMP into the bacterial chromosome is unavoidable.

DUT and UNG are two important genes that are common to many species as they have important functions in the organisms. Mutated DUT is responsible of enhancing incorporation rate of uracil into dsDNA and UNG has a function to cut heavily rich Uracilated DNA. These properties are important for us as we can create what we have coined Uplasmid which enable correct expression of the genes like in a classical plasmid but having gained the property to prevent its exchange with surrounding organisms.

In organisms with double mutations, the DNA contains a significantly higher level of uracil. Surprisingly, these cells are able to survive, likely because most of the uracil units in this context actually function as substitutes for thymine. This substitution maintains the same coding characteristics as the Watson-Crick base pairing.

As explained on the engineering page, we devised an innovative method to produce the so coined term Uplasmid through a bacterial strain named CJ236 that has both mutations.

Uplasmid : benefits and testing

We showed that Uplasmids have the ability to still express the protein it encodes while inserted into a strain that has at least the UNG gene mutation.

Here are the results (see more on results page).

We have showed that we prevented more than 90% of the cases of HGT. dut+/ung+ transformed cells were mostly non viable on the plates.

More information on the engineering page.