Safety
According to the World Health Organization “Laboratory biosafety” consists of the containment principles, technologies and practices that are implemented to prevent unintentional exposure to pathogens and toxins, or their accidental release.
Before starting to work in the lab, team members have received biossecurity training and learned the correct procedures to do the experiments safely. The team used three laboratories to execute lab work: Laboratório de Biotecnologia Molecular - LBM (Biosafety level 2), SynBiom (Biosafety level 1), and Laboratório de Biologia Molecular e Computacional de Fungos - LBMCF (Biosafety level 1) in different universties: Universidade Federal de Viçosa and Universidade Federal de Minas Gerais. However, the headquarters laboratories were LBM and Synbiom.
Lab 1 - UFV Level 2:
Lab 2 - UFMG Level 1:
Lab 3 - UFMG Level 1:
Safety norms that were strictly followed were:
The experiments conducted in the development of “ProChi” used bacteria strains that are non-pathogenic: E. coli (DH5α, BL21) and Lactobacillus acidophilus. We also used only non-hazardous parts, which cause no biosafety risk.
Bearing in mind the importance of thinking about our project from a biosafety perspective, we developed a kill switch design based on two mechanisms of action: cell death and the infeasibility of heterologous parts (exochitinase and endochitinase).
To develop the circuit, we took into account inputs common to the external environment and the physiological environment of the human gut but dependent on a gradient as a form of activation. In this sense, we chose two promoters inducible by temperature and hypoxia respectively: lambda cI promoter (BBa_R0051) and pfdhF promoter (BBa_K387003). The lambda cI promoter is activated when the temperature is greater than or equal to 35º C due to the denaturation of a repressor protein that has an affinity for the promoter: cIts (BBa_K098997). In turn, the pfdhF promoter is inducible by an oxygen-affinity transcription factor fdhA which binds to the pfdhF promoter under hypoxia conditions, initiating transcription.
Both temperature and hypoxia conditions were the activation parameters chosen due to the physiological temperature of the human body and the condition of low oxygen concentration in the intestinal lumen.
In the image above, we have the temperature-dependent circuit in its activated condition. Note that the denatured protein (in yellow) cannot repress the lambda cI promoter (in blue). As a result, transcription of PhlF is initiated and its product represses the PhlF repressible promoter (green arrow). This prevents transcription of Cre recombinase, a protein-dependent on the Loxp site to invert a DNA sequence. In this specific case, the absence of Cre prevents the inversion of the endochitinase and exochitinase promoters.
The image above shows the kill switch device active due to temperature reduction (below 35ºC). This prevents transcription of the lambda repressor, which prevents repression of the PhlF repressible promoter (green arrow). This promotes Cre transcription and inversion of the endochitinase and exochitinase promoters. This construct was developed to prevent the cross-exchange of genetic material between transformed and untransformed bacteria.
The second kill switch construct aims at cell death through endolysin. LysKB317 (BBa_K4133012) is a specific and selective toxin that affects Lactobacillus. Its transcription promotes the cell death of our chassi.
In the image above, we have a constitutive promoter transcribing fdhA(BBa_K1878004). We also have the hypoxia-inducible pfdhF promoter (BBa_K387003). FdhA is a protein that binds to the pfdhF promoter but has an affinity for oxygen. Under hypoxic conditions it maintains a preference for the FdhA-dependent promoter. In this condition, there is the expression of SrpR, a repressor of the SrpR repressible promoter (red arrow) (BBa_K1725020). In this context, there is inhibition of endotoxin.
In a regular atmospheric environment, oxygen competes for dfhA, repressing the hypoxia-sensitive promoter. This repression, in turn, induces endolysin transcription, killing the cell.
In the future, we intend to add the heterologous genes to the chromosomal DNA to avoid escape by bacterial conjugation. We also intend to characterize Kill Switch parts in next steps. The constructs have been added to the registry.
To reduce risks, we have decided to test our concept with the organism C. elegans, a model organism for nematoids, instead of using pathogenic worms such as Ascaris lumbricoides.
The contaminated residues were discarded in autoclavable bags and when full, they were autoclaved, disposal of supernatants and liquid solutions, in general, was carried out in wide-mouth autoclavable flasks containing absorbent paper soaked in disinfectant solution at the bottom of the flask. After that, they were discarded into infectious waste. The infected sharp materials, such as pipette tips, were disposed of in previously named leak-resistant containers. When they were three-quarters full, they were autoclaved and discarded in infectious waste.
The inactivation and disposal protocol that we used followed Resolution Nº 2, of November 27, 2006, established by the Brazilian National Technical Commission on Biosafety – CTNBio, in compliance with the precepts of LAW 11.105, of March 24, 2005. This Law. regulate items II, IV, and V of § 1º of art. 225 of the Federal Constitution, and establishes safety standards and inspection controls for activities that control genetically modified organisms - GMOs and their results.
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LU, S.Y.; et al. Recombinant bacteriophage LysKB317 endolysin mitigates Lactobacillus infection of corn mash fermentations. Biotechnology for Biofuels, 13(1), 2020. https://doi.org/10.1186/s13068-020-01795-9