S a f e t y

Protocols

All experiments are conducted in biosafety level 1 laboratories at the CNRS LISM premises in Marseille, under the supervision of qualified teacher-researchers. This level of containment is appropriate for handling organisms that pose minimal risk to health and the environment. All team members underwent training in laboratory safety rules at the start of the project, including: good practices for handling biological materials, management of biological and chemical waste, prevention of chemical risks such as the handling of reagents such as acrylamide, SDS, and TEMED, etc. And on the use of equipment such as fume hoods, centrifuges, Bunsen burners, and UV sources. Each handling operation is carried out in accordance with internal procedures, with the systematic use of appropriate personal protective equipment (PPE) such as lab coats, gloves, safety glasses, etc. The laboratory is equipped with safety devices such as eye wash stations, fire extinguishers, and first aid kits, and health and safety officers are available to answer any questions we may have.

Equipment

Figure 1: Laboratory workbench

Figure 2: Microbiology work area

Figure 3: Laminar flow hood

Organism Selection

Organism Selection: We chose to use well-characterized strains of Escherichia coli: DH5α, BL21, CC118, and W3110, all classified as risk level 1. These strains are non-pathogenic, do not produce toxins, and lack virulence factors, and are widely used in molecular biology. In our project, these organisms are modified to express melanin biosynthesis pathways using genes encoding enzymes such as tyrosinase tyr1 and a mutation on the aroF gene, which encodes a protein involved in the shikimate pathway of melanin synthesis, thereby increasing melanin production. This E. coli organism is classified as risk level 1, which means it poses a low health risk. We therefore consider the risks to be low. However, in order to ensure maximum safety by limiting horizontal gene transfer and preventing leaks into the environment, we have designed an auxotrophy for our genetically modified bacteria. We have modified our chassis to add a deletion of the murI gene, which codes for racemase, enabling the synthesis of DL glutamate, an amino acid necessary for the formation of peptidoglycan. This deletion makes bacterial growth dependent on a supply of DL-glutamate, which prevents any proliferation outside a controlled environment. Growth tests on media with different concentrations of DL glutamate have confirmed the effectiveness of this strategy.

Containment System

In addition to auxotrophy, we have chosen to add physical containment by encapsulating our modified bacteria in an alginate-based hydrogel. This material forms a protective matrix that limits bacterial dispersion into the external environment. This addition allows us to guarantee, from a theoretical standpoint, patient safety during treatment injection. We also wish to modify certain components of the hydrogel to minimize potential risks and enhance its containment properties and in vivo stability, reduce the risk of premature degradation, and improve its mechanical and barrier properties.

Biosafety testing

Our genetically modified strains will undergo rigorous biosafety testing in accordance with the requirements of the competent authorities. These tests are designed to assess the genetic stability of the modifications, the absence of dissemination into the environment, and safety for users and patients. We are committed to complying with all applicable laws and regulations, in particular those governing the use of GMOs, such as European Directive 2009/41/EC on the contained use of GMOs, the recommendations of the High Council for Biotechnology (HCB), and the biosafety standards of the CNRS and the university campus. No animal or human experimentation is planned as part of this project, and no clinical or agricultural applications are envisaged at this stage.