Biosafety issues of the organisms
The organisms we are dealing with are L. lactis, and E. coli DH5α. L. lactis is general regarded as safe (GRAS) and has s long history in food fermentation and preservation, while E. coli is in Risk Group 1 and can not cause disease in healthy adults. L. lactis is used to produce cheese. It is also record in use as probiotic bacterium for it can secreting health beneficial extra cellular polysaccharide and other metabolites and macromolecules. A recombinant L. lactis expressing the IL-10 to treat Crohn disease has been approved by the USA FDA. E. coli is the most studied and utilized bacteria in synthetic biology. Professor Yanguang Cong kindly provided us with the genomic DNA of a virulence attenuated S.Typhi vaccine, Ty21a. The Vi antigen locus DNA fragment was produced using PCR, taking the S.Typhi genomic DNA as the template. We didn’t culture S.thphi in the lab. The pNisZ promoter sequences, the lacZ gene we utilized, the nisI gene, the nsr gene and other DNA fragments we used in this study are derived from other GRAS organisms.
Biosafety considerations of antibiotics
Another biosafety issue we considered and resolved in our project is the antibiotic resistant genes. Due to the overuse and inappropriate application of antibiotics in medical treatment, in animal farming and other practices, multiple antibiotic resistant bacteria have rapidly evolved. In our design, the recombinant L. lactis is devoid of any antibiotic resistant genes. In the lab, the supplemented antibiotics in culture medium such as ampicillin and erymycin are strictly treated according to the rules in the lab. The nisin is a food grade antibacterial peptide and has been widely used in food preservation. To reduce the potential risk of releasing nisin into the environment to influence ecological niches, the nisin supplemented culture medium is heated, and the nisin is denatured and antibacterial activity is lost.
Horizontal gene transfer possibilities and biological containment
The genome integrated DNA fragments are localized at the His locus. At this locus, no IS sequences, transposons and other mobile DNA elements were present. In other words the His locus is refractory to horizontal transfer. What is more, the His locus is required for the biosynthesis of histidine which is essential for the growth of L. lactis. Without histidine supplement in the culture, the His locus targeted device knocked in recombinant L. lactis can only be replicated in the lab. The His locus disrupted recombinant L. lactis strains can not survive in the wild for histine is very limited in the environment. By this way, the recombinant L. lactis strains are biologically contained in the lab. A similar biological containment strategy has been applied to the IL-10 recombinant L. lactis using the thyA locus. The thyA locus is required for the synthesis of thymidine, which is also essential for the growth of L. lactis.
Biosafety training and protection actions in the lab
The experiments are mainly performed at the molecular level. Some biosafety risks may be caused by the reagents we used. For example, we need to use DNA staining molecules for visualization after electrophoresis. All of us wear gloves when dealing with DNA staining molecules. The biological or chemical wastes were collected and sterilized, and the wastes were further treated by professional staffs in our department. Before our research, all of us have taken an online biosafety training course. Furthermore, we received one week biosafety training in the lab. We learned how to operate the UV light-equipped biosafety hood and the high pressure vapour sterilizer. We can clearly discriminate the contaminated area by DNA staining molecules from other clean area. The lab we carried out our experiments is a biosafety level 2 lab according to local regulation agencies. All the members strictly followed the guideline and rules to handle and perform experiments. We wear personal protective equipment, such as lab coats and lab gloves to protect us.