Introduction
Personal and environmental safety must be the main concern of any synthetic biology project, and that is not open for discussion. Therefore, the BioPank project has given importance to biosafety and biosecurity since the beginning of the project development.
SynFronteras has a multidisciplinary team oriented to risks involving the engineering and handling of GMOs. All members of the drylab and wetlab group took a course on bioethics and biosafety in the academic curriculum and have experience in handling microorganisms.
Laboratories
Access:
Access to laboratories is extremely controlled. First, access to the campus is controlled by the institutional security of the university, not allowing the entry of people not linked to the university and students without authorization to access the campus. The research laboratory used has access only through biometric recognition, which records the date and time of the user's entry into the laboratory. The teaching laboratories, which were mostly used to prepare solutions, were accessed under the supervision of the technicians responsible for these laboratories, who helped in the operation of the equipment and in the management of waste.
Personal Protection
The use of PPE is mandatory throughout the laboratory environment. During the entire process of handling the microorganisms, a cotton lab coat, gloves, closed shoes and pants that fully cover the legs were used. In addition, for handling leishmania, disposable coats were used, discarded in biological waste.
Due to the COVID-19 pandemic, the number of laboratory users is limited and, in addition, masks are a mandatory protective item, preventing the spread of SARS-CoV-2.
The entire process of handling microorganisms was carried out using a level II biological safety cabinet, with a flame, ideal for containing the spread of microorganisms into the environment.
The laboratory also has a gas exhaust hood for handling volatile substances. All equipment has a usage protocol attached to it or available in the laboratory protocol book, which helps to avoid accidents due to misuse.
Waste management
All material that has been in contact with microorganisms or other contaminated materials has been autoclaved and properly disposed of in biological waste.
The few chemical residues generated were destined for the university's chemical waste disposal process.
Project Biosecurity
Strains
For the project steps already carried out, the strains E. coli DH10B, DH5-alpha, BL21 (D3) were used, all classified in risk group 1.
Leishmania, for the leishmanicidal test, was cultivated under the responsibility and guidance of a researcher with extensive experience with this microorganism and all tests were carried out in a level II biological safety cabinet with all the necessary precautions and protective equipment.
OGM and Legislation
Although the ultimate goal of the project is the release of the genetically modified bacteria into the environment, this step will not be carried out. So far, only AMP production validation tests and leishmanicidal action have been performed in vitro, using E. coli. The process until the release of the genetically modified bacteria into the environment is quite extensive, requiring a rigorous validation process and a long, bureaucratic and unknown regulatory path with the government, mainly because Brazilian legislation is not clear about the regulatory process for microorganisms destined for paratransgenesis, which will demand extensive legal assistance, unattainable in the short term.
Risks and Biocontainment mechanisms
In order to provide security for the future application of this strategy of paratransgenesis to control leishmaniasis, several considerations were carried out and sundry safety mechanisms were designed.
First, the engineered bacterium's leishmanicidal action is based on the production of AMPs. AMPs are widely found in nature, produced naturally by numerous prokaryotic and eukaryotic species and, in general, do not pose risks to humans and the environment.
The first security mechanism to be thought of for this project was the implementation of a kill-switch, which is one of the main biosecurity practices in synthetic biology projects. A kill-switch mechanism is designed to prevent the genetically modified bacteria from spreading through the environment. Our kill-switch is activated when the bacteria is exposed to light, promoting their death in the process. However, we have not yet reached the testing phase to verify the effectiveness of this kill-switch.
Another safety mechanism designed for the project is that the bacteria were designed to produce AMP only within Lutzomyia longipalpis. For this, a toehold RNA switch mechanism was used, activated by a miRNA found specifically in the midgut of the sandfly.