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| - | <h1 id="h1_lf" class="main_tit"><div>Safety</div></h1>
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| - | <h1 id="h1_rt" class="main_tit"><div>More</div></h1>
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| - | <div id="box_main"> <!-- start box_main -->
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| - | <div class="box_contenuti">
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| - | <ol>
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| - | <li>
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| - | <span>Would any of your project ideas raise safety issues in terms of:</span>
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| - | <ul>
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| - | <li>researcher safety,</li>
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| - | <li>public safety, or</li>
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| - | <li>environmental safety</li>
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| - | </ul>
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| - | <p>
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| - | Before starting working in the lab we were trained by
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| - | biosafety experts about the rules to follow in laboratory.
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| - | </p>
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| - | <span>The key points are:</span>
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| - | <ul>
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| - | <li>
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| - | the general behavior rules we must follow, like wearing
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| - | gloves and lab coat when we manage chemicals and cellular
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| - | cultures, never eating or drinking inside the work area
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| - | and smoking outside the building;
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| - | </li> <li>
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| - | the equipment we need to use, as the UV transilluminator,
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| - | the laminar flow cabinets, the biosafety hoods, the confocal
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| - | microscope and the autoclave for tubes and glassware used
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| - | for cellular cultures and bacteria;
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| - | </li> <li>
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| - | the containment and the waste handling procedures, in order
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| - | to avoid the involuntary spreading of microorganisms;
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| - | </li> <li>
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| - | the waste handling procedures, in order to avoid the
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| - | involuntary spreading of toxic substances;
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| - | </li>
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| - | </ul>
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| - | <p>
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| - | We planned a risk evaluation based on the Italian and UE guidelines and we concluded that
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| - | our project does not represent a danger for public and environmental safety. We performed
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| - | all experiments according to the protocols that respect the current regulations in Italy.
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| - | (for the current laws about researcher, public and environmental safety in Italy click here,
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| - | Dlgs 206/01, Dlgs 81/08 and DM 25.09.2001 )
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| - | </p>
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| - | <p>
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| - | Regarding the biological part of the project, we used only harmless bacteria strains incapable
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| - | to survive outside the laboratory environment like the E.coli strain DH5-alpha, strain HB2151
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| - | and strain W3110. We used heat and Sodium hypochlorite to kill all bacterial cultures at the end
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| - | of the experiments. We also used E.coli strain Nissle, that is able to live in the external
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| - | environment but it is regularly used as a probiotic for many years, so it is dangerous for the
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| - | human safety.
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| - | </p>
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| - | <p>
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| - | The genetic material we used in our project was not extracted directly from other prokaryotic
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| - | or eukaryotic species. All new sequences that can not be found as biobricks, were synthesized.
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| - | </p>
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| - | <p>
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| - | Regarding the antibodies, instead of real virions we used virus like particles (VLP) to test
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| - | our antibodies. Viral like particles are viral protein envelops that do not contain a viral
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| - | genome so they are non-infectious.
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| - | </p>
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| - | <span>Both the toxins we intend to use in the project are safe:</span>
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| - | <ol>
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| - | <li>
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| - | Our first choice is Cathelicidin LL-37 associated with Holin. LL-37 is a human
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| - | antimicrobial peptide, not dangerous to humans nor the environment. Holin is a small
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| - | bacteriophage-encoded protein that accumulate in the membrane until, at a precise genetically
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| - | programmed time, the membrane suddenly becomes permealized.
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| - | </li>
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| | | | |
| - | <li>
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| - | Alternatively, we can use Tse2, a P.aeruginosa toxin which blocks the growth of
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| - | prokaryotic and eukaryotic cells when expressed intracellularly, but the secreted toxin does
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| - | not have effect on eukaryotic cells.
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| - | </li>
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| - | </ol>
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| - | <p>
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| - | Moreover, our project was implemented with different strong security systems that allow the
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| - | total control over the molecular platform avoiding the horizontal gene transfer.
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| - | </p>
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| - | </li>
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| - | <li>
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| - | <strong>Do any of the new BioBrick parts (or devices) that you made this year raise any safety
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| - | issues? If yes,
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| - | <ol>
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| - | <li>did you document these issues in the Registry?</li>
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| - | <li>how did you manage to handle the safety issue?</li>
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| - | <li>How could other teams learn from your experience?</li>
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| - | </ol>
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| - | </strong>
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| - | <p>
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| - | The biobricks we made, do not produce protein with toxic effects on humans, plants and animals. The
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| - | cathelicidin LL-37 represents a threat for prokaryotes only.
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| - | </p>
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| - | </li>
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| - |
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| - | <li>
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| - | <strong>
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| - | Is there a local biosafety group, committee, or review board at your institution?
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| - | <ul>
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| - | <li>If yes, what does your local biosafety group think about your project?</li>
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| - | <li>If no, which specific biosafety rules or guidelines do you have to consider in your country?</li>
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| - | </ul>
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| - | </strong>
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| - |
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| - | <p>
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| - | The ICGEB (International Centre for Genetic Engineering and Biotechnology) has a Biosafety Unit
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| - | that is especially focused on the effect of the new technologies on the environment, according to
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| - | the Italian laws (click here to learn more about the ICGEB goals in this field). We explained to
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| - | them our project in detail and then we also filled a form about genetically modified organisms
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| - | requested from the Italian government to estimate the biosafety risks. The ICGEB Biosafety
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| - | Unit evaluated our project and they did not find any particular danger concerning the safety
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| - | and security beyond the usual.
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| - | </p>
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| - | <span>We quote you the summary of the form about genetically modified organisms:</span>
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| - | <p>
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| - | “We used the Escherichia coli ( Bacteria / Eubacteria / Proteobacteria / Gammaproteobacteria/
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| - | Entero-bacteriales / Enterobacteriaceae / Escherichia / E.coli ) strain Nissle 1917, commercialized
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| - | as Mutaflor, as the receiving organism. Mutaflor is commonly used as a probiotic and it can
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| - | be found in the German Collection of Microorganisms in Braunschweig. Several clinical trials
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| - | demonstrate that this strain is harmless to humans and that it has many beneficial effects.
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| - | E. coli Nissle 1917, was isolated by A. Nissle in 1917 from the feces of a soldier who, in
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| - | contrast with all of his comrades, did not develop enterocolitis during the war on the Balkan
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| - | peninsula, which was highly contaminated by enteropathogens at that time.
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| - | We confirmed its identity using standard microbiology techniques and sequencing the following
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| - | genes 16S, rpoB, recA . This particular strain is "06:K5:H1": it has an iron uptake system, type
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| - | I fimbriae, cryptic plasmids and semirough LPS. It produces microcines, but no proteic toxins are
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| - | produced, so it doesn't present any danger to animals or plants. It does not contain mycoplasma,
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| - | viruses or viroids, and doesn't have any biogeochemical properties. Genetic instability or any
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| - | previous genetic modification are not reported. E. coli Nissle 1917 is a ubiquitous strain found
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| - | in all countries. It lives in the ground, in animal or human intestines as a commensal where
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| - | it competes with other bacteria. This bacterium does not form spores on any other quiescent form.
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| - | </p>
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| - | <p>
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| - | We transformed the E.coli strain Nissle 1917 with a protein-expressing plasmid J61002 ( 2267
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| - | bp). We decided to insert a gene into its genome to prevent the horizontal gene transfer and
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| - | to keep under control the bacterial growth. Another plasmid in combination with a transposase
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| - | coding plasmid was used to integrate two copies of CymR regulator into the chromosome downstream
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| - | GLMS gene. The integration does not interrupt other genes.
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| - | </p>
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| - | <span>Main features of the vector used:</span>
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| - | <ul>
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| - | <li>Structural genes: Antibody (SIP/scFv) fused to LPP-OmpA</li>
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| - | <li>Antibiotic resistant</li>
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| - | <li>marker gene: Ampicillin</li>
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| - | <li>Other genes: Tse2 and LL-37 coding genes</li>
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| - | <li>Regulatory elements: Consitutive promoter and Cumate inducible promoter repressed by CymR and induced by Cumate</li>
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| - | <li>Sites used for the insertion: EcoRI and PstI</li>
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| - | <li>ORI: ColE1</li>
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| - | <li>Mobility: not mobilizable</li>
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| - | <li>Copy number: high copy (50-70 copies/cell)</li>
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| - | </ul>
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| - |
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| - | <p>
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| - | The sequences that we inserted are not pathogenic or toxic to animals or plants. In our project
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| - | we use specific tissue cDNA, synthetic DNA and biobricks.
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| - | </p>
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| - | <span>The genetic elements that we used:</span>
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| - | <ul>
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| - | <li>REPRESSOR - to control the toxin expression</li>
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| - | <li>TOXIN - to control both the dimension of the bacterial population and to prevent eventual plasmid transfer to other bacteria</li>
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| - | <li>LPP-OmpA - signal to express the protein fused to the membrane</li>
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| - | <li>scFv/SIP - single chain variable fragment antibodies to capture the antigens</li>
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| - | </ul>
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| - | <p>
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| - | All the constructs made were fully sequenced and they do not contain any unknown sequence or
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| - | sequence codifying for undesired functions. The transcripts are the ones that we expected as
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| - | their proteins product. Like the original Nissle, our bacterium is not pathogenic and grows
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| - | less well than the wild-type strain. It also has the same environmental impact as the wild type,
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| - | and it is very stable against mutation even after 50 generations. There are no possibilities
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| - | of horizontal transfer thanks to the addition of a safety gene guard system. The presence of
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| - | pJ61002 plasmid confers to our strain Ampicillin resistance. This kind of marker will be used
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| - | only during the laboratory experiments a then will be replaced with a biocompatible marker.
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| - | Our project may contemplate the inoculation into animals (mice) in the future.
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| - | </p>
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| - | <p>
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| - | For our experiments we utilized a maximum of 50mL of culture. The preparation of the cultures
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| - | consists in inoculating the bacteria in solid or in liquid medium. The bacterial culture can
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| - | reach the highest concentration of 7x10^9 bacteria per ml. All the equipment and the glassware
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| - | used in the laboratory during the project, have been autoclavated and sterilized after use.“
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| - | Vittorio Venturi
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| - | </p>
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| - | <p>
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| - | This domument was approved by Vittorio Venturi (a legal responsible about safety in the
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| - | laboratories where the team is working in) and Marco Vegliach (a safety assistant)
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| - | </p>
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| - | </li>
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| - | <li>
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| - | <strong>
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| - | Do you have any other ideas how to deal with safety issues that could be useful for
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| - | future iGEM competitions? How could parts, devices and systems be made even safer through
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| - | biosafety engineering?
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| - | </strong>
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| - |
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| - | <p>
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| - | Our advice for the future iGEM competitions is to avoid harmful substances like chloramphenicol or
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| - | ethidium bromid during the experiments. Ethidium bromid is an intercalating agent commonly used
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| - | as a nucleic acid stain for agarose gel electrophoresis. It is also a known mutagen. Ethidium
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| - | bromide can be replaced with a non toxic nucleid acid stain like Syber green. Chloramphenicol
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| - | is broad-spectrum antibiotic used as selection marker in standard iGEM vectors. This antibiotic
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| - | has serious adverse effects like bone marrow toxicity so it should be replaced with a less
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| - | toxic marker. Our advise to iGEM is to create a safety regulations that all teams should
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| - | follow. In this way iGEM can achieve a higher level of biosafety control.
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| - | </p>
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| - | </li>
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| - | </ol>
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| - | <h2>Contact us</h2>
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| - | <p>For other information, write to:</p>
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