Before starting working in the lab we were trained by biosafety experts about the rules to follow in laboratory.

• the general behavior rules we must follow, like wearing gloves and lab coat when we manage chemicals and cellular cultures, never eating or drinking inside the work area and smoking outside the building;
• the equipment we need to use, as the UV transilluminator, the laminar flow cabinets, the biosafety hoods, the confocal microscope and the autoclave for tubes and glassware used for cellular cultures and bacteria;
• the containment and the waste handling procedures, in order to avoid the involuntary spreading of microorganisms;
• the waste handling procedures, in order to avoid the involuntary spreading of toxic substances;

We planned a risk evaluation based on the Italian and UE guidelines and we concluded that our project does not represent a danger for public and environmental safety. We performed all experiments according to the protocols that respect the current regulations in Italy. (for the current laws about researcher, public and environmental safety in Italy click here, Dlgs 206/01, Dlgs 81/08 and DM 25.09.2001 )

Regarding the biological part of the project, we used only harmless bacteria strains incapable to survive outside the laboratory environment like the E.coli strain DH5-alpha, strain HB2151 and strain W3110. We used heat and Sodium hypochlorite to kill all bacterial cultures at the end of the experiments. We also used E.coli strain Nissle, that is able to live in the external environment but it is regularly used as a probiotic for many years, so it is not dangerous for the human safety.

The genetic material we used in our project was not extracted directly from other prokaryotic or eukaryotic species. All new sequences that can not be found as biobricks, were synthesized.

Regarding the antibodies, instead of real virions we used virus like particles (VLP) to test our antibodies. Viral like particles are viral protein envelops that do not contain a viral genome so they are non-infectious.

1. Our first choice is Cathelicidin LL-37 associated with Holin. LL-37 is a human antimicrobial peptide, not dangerous to humans nor the environment. Holin is a small bacteriophage-encoded protein that accumulate in the membrane until, at a precise genetically programmed time, the membrane suddenly becomes permealized.
2. Alternatively, we can use Tse2, a P.aeruginosa toxin which blocks the growth of prokaryotic and eukaryotic cells when expressed intracellularly, but the secreted toxin does not have effect on eukaryotic cells.

Moreover, our project was implemented with different strong security systems that allow the total control over the molecular platform avoiding the horizontal gene transfer.

The biobricks we made, do not produce protein with toxic effects on humans, plants and animals. The cathelicidin LL-37 represents a threat for prokaryotes only.

The ICGEB (International Centre for Genetic Engineering and Biotechnology) has a Biosafety Unit that is especially focused on the effect of the new technologies on the environment, according to the Italian laws (click here to learn more about the ICGEB goals in this field). We explained to them our project in detail and then we also filled a form about genetically modified organisms requested from the Italian government to estimate the biosafety risks. The ICGEB Biosafety Unit evaluated our project and they did not find any particular danger concerning the safety and security beyond the usual.

“We used the Escherichia coli ( Bacteria / Eubacteria / Proteobacteria / Gammaproteobacteria/ Entero-bacteriales / Enterobacteriaceae / Escherichia / E.coli ) strain Nissle 1917, commercialized as Mutaflor, as the receiving organism. Mutaflor is commonly used as a probiotic and it can be found in the German Collection of Microorganisms in Braunschweig. Several clinical trials demonstrate that this strain is harmless to humans and that it has many beneficial effects. E. coli Nissle 1917, was isolated by A. Nissle in 1917 from the feces of a soldier who, in contrast with all of his comrades, did not develop enterocolitis during the war on the Balkan peninsula, which was highly contaminated by enteropathogens at that time. We confirmed its identity using standard microbiology techniques and sequencing the following genes 16S, rpoB, recA . This particular strain is "06:K5:H1": it has an iron uptake system, type I fimbriae, cryptic plasmids and semirough LPS. It produces microcines, but no proteic toxins are produced, so it doesn't present any danger to animals or plants. It does not contain mycoplasma, viruses or viroids, and doesn't have any biogeochemical properties. Genetic instability or any previous genetic modification are not reported. E. coli Nissle 1917 is a ubiquitous strain found in all countries. It lives in the ground, in animal or human intestines as a commensal where it competes with other bacteria. This bacterium does not form spores on any other quiescent form.

We transformed the E.coli strain Nissle 1917 with a protein-expressing plasmid J61002 ( 2267 bp). We decided to insert a gene into its genome to prevent the horizontal gene transfer and to keep under control the bacterial growth. Another plasmid in combination with a transposase coding plasmid was used to integrate two copies of CymR regulator into the chromosome downstream GLMS gene. The integration does not interrupt other genes.

• Structural genes: Antibody (SIP/scFv) fused to LPP-OmpA
• Antibiotic resistant
• marker gene: Ampicillin
• Other genes: Tse2 and LL-37 coding genes
• Regulatory elements: Consitutive promoter and Cumate inducible promoter repressed by CymR and induced by Cumate
• Sites used for the insertion: EcoRI and PstI
• ORI: ColE1
• Mobility: not mobilizable
• Copy number: high copy (50-70 copies/cell)

The sequences that we inserted are not pathogenic or toxic to animals or plants. In our project we use specific tissue cDNA, synthetic DNA and biobricks.

• REPRESSOR - to control the toxin expression
• TOXIN - to control both the dimension of the bacterial population and to prevent eventual plasmid transfer to other bacteria
• LPP-OmpA - signal to express the protein fused to the membrane
• scFv/SIP - single chain variable fragment antibodies to capture the antigens

All the constructs made were fully sequenced and they do not contain any unknown sequence or sequence codifying for undesired functions. The transcripts are the ones that we expected as their proteins product. Like the original Nissle, our bacterium is not pathogenic and grows less well than the wild-type strain. It also has the same environmental impact as the wild type, and it is very stable against mutation even after 50 generations. There are no possibilities of horizontal transfer thanks to the addition of a safety gene guard system. The presence of pJ61002 plasmid confers to our strain Ampicillin resistance. This kind of marker will be used only during the laboratory experiments a then will be replaced with a biocompatible marker. Our project may contemplate the inoculation into animals (mice) in the future.

For our experiments we utilized a maximum of 50mL of culture. The preparation of the cultures consists in inoculating the bacteria in solid or in liquid medium. The bacterial culture can reach the highest concentration of 7x10^9 bacteria per ml. All the equipment and the glassware used in the laboratory during the project, have been autoclavated and sterilized after use.“ Vittorio Venturi

This domument was approved by Vittorio Venturi (a legal responsible about safety in the laboratories where the team is working in) and Marco Vegliach (a safety assistant)

Our advice for the future iGEM competitions is to avoid harmful substances like chloramphenicol or ethidium bromid during the experiments. Ethidium bromid is an intercalating agent commonly used as a nucleic acid stain for agarose gel electrophoresis. It is also a known mutagen. Ethidium bromide can be replaced with a non toxic nucleid acid stain like Syber green. Chloramphenicol is broad-spectrum antibiotic used as selection marker in standard iGEM vectors. This antibiotic has serious adverse effects like bone marrow toxicity so it should be replaced with a less toxic marker. Our advise to iGEM is to create a safety regulations that all teams should follow. In this way iGEM can achieve a higher level of biosafety control.