Difference between revisions of "Team:Toulouse-INSA-UPS/Results"

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          <h1>SAFETY</h1>
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<h1 class="heavy">RESULTS</h1>
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        <hr/>
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<p>
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The whole Cerberus project is based on the construction and validation of a proteic platform to link very different molecules together. This platform is composed of three heads. The first head is a CBM3a domain which specifically bind cellulose. The second head is a streptavidin domain with a strong affinity to biotinylated compounds. The third head is based on the presence of an unnatural amino acid to covalently link any molecule with an alkyne moiety.
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</p>
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<p>To validate the Cerberus platform, we worked head by head, starting from the CBM3a validation, then the streptavidin domain assessment and finally the demonstration of the unnatural amino acid capacity to covalently bound alkynilated molecules. Thus, our first construction has only one head and was named Sirius, after the brightest star of the northern hemisphere alpha canis majoris. The second construction presents both the CBM3a and streptavidin heads and was nicknamed Orthos, as a two-headed mythological dog. For sure, the final construction is Cerberus and its three heads.</p>
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<div id="HP_Tab_Group">
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        <p>There is a lot of dangers in a laboratory. So, everything cannot be done and there are rules to respect to protect the environment and ourselves. Due to an atmosphere of mistrust concerning GMOs in France, it is also very important to us to appease the opinion with a strict safety policy.</p>
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        <p>First, we will present here how the safety department in INSA is organized: what kind of structure received us, what is the French legislation concerning risks and how we have been formed. Then, we will detail what kind of risks we could met during the lab work and how we should avoid them with good laboratory habits. Finely, we will explain how we constructed our project to minimize the health and safety issues.</p>
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<a class="nav-link btn btn-secondary rounded-top corner-bottom mr-1 active" data-toggle="pill" href="#Results_Part1" role="tab">Sirius: CBM3a</a>
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</li>
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<li class="nav-item">
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<a class="nav-link btn btn-secondary rounded-top corner-bottom mr-1" data-toggle="pill" href="#Results_Part2" role="tab">Orthos: Streptavidin</a>
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<a class="nav-link btn btn-secondary rounded-top corner-bottom mr-1" data-toggle="pill" href="#Results_Part3" role="tab">Cerberus: AzF</a>
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<a class="nav-link btn btn-secondary rounded-top corner-bottom mr-1" data-toggle="pill" href="#Results_Part4" role="tab">Bacterial Cellulose</a>
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        <div id="Description_tab">
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                        <a class="nav-link btn btn-secondary rounded-top corner-bottom mr-1 active" data-toggle="pill" href="#Safe_Part1" role="tab">Safety Department</a>
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                        <a class="nav-link btn btn-secondary rounded-top corner-bottom mr-1" data-toggle="pill" href="#Safe_Part2" role="tab">Knowing the Risks</a>
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                    </li>
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<h2 id="Sirius" class="heavy">Sirius: CBM3a</h2>
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                        <a class="nav-link btn btn-secondary rounded-top corner-bottom mr-1" data-toggle="pill" href="#Safe_Part3" role="tab">Project Construction</a>
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                    </li>
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<h3 id="Background_Sirius" class="heavy">Background</h3>
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              </ul>
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                    <p>
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Sirius is a fusion protein between CBM3a and mRFP1. This design, which consists in fusing CBM3a to a fluorescent moiety, allowed us to investigate the binding capability of the CBM3a domain to cellulose through the four aromatic amino acids of its binding domain. mRFP1 was chosen since it is both a coloured and fluorescent protein.
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</p>
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                    <h3 id="Key_Achievements_Sirius" class="heavy">Key Achievements</h3>
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                    <!--PART 1 -->
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                            <h2 id="SD" class="heavy">INSA Safety Department</h2><hr/>
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<div style="text-align:center">
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                            <figure class="figure"  style="text-align:center;">
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                                <img style="width : 70%; heigth = auto;" src="https://static.igem.org/mediawiki/2018/d/d3/T--Toulouse-INSA-UPS--BET-room.jpg" class="figure-img img-fluid rounded" alt="A generic square placeholder image with rounded corners in a figure.">
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                                <figcaption class="figure-caption"><strong>Figure 1:</strong> Ethidium Bromide Handling Room.</p>
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                                    </figcaption>
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                                    </figure>
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</div>
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                          <h3 id="SD-org" class="heavy">Organization of the INSA Safety Department</h3>
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                            <p>We have been received by the LISBP. The head of the laboratory where we worked is Carole Jouve and the organization of the safety department is described in the figure 1 below. We are very grateful for the welcoming of our colleagues and the time they spend to form us.</p>
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<div style="text-align:center">
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<figure class="figure"  style="text-align:center;">
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                                <img style="width : 80%; heigth = auto;" src="https://static.igem.org/mediawiki/2018/b/b1/T--Toulouse-INSA-UPS--All--Angeline--Safety1.png" class="figure-img img-fluid rounded" alt="organization chart">
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                                <figcaption class="figure-caption"><i><strong>Figure 2:</strong> Organization chart of the INSA safety department.</i>
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                                    </figcaption>
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                                    </figure>
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</div>
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                          <h3 id="SD-law" class="heavy">Legislation and French labor Law</h3>
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                            <p>We are working in a french engineering school (INSA Toulouse), thus we have to respect the french national regulations about working conditions and manipulation of GMOs.</p>
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                            <p>As we work with microorganisms, we are concerned by the regulation on workers protection against risks resulting from their exposure to pathogenic biological agents (Decree No. 94-352 of 4 May 1994). It also includes human endoparasites which may cause infections, allergies or toxicity. This Decree is the French transposition of the Directive 90/679 / EEC and is also transcribed in the Labour Code (Articles L4421-1 R4421-1 to R4427-5). This Decree of the 16th July 2007 describes the technical preventive measures that must be followed in research laboratories, where workers are likely to be exposed to biological pathogens. We must obey to the rules of health, safety, and preventive medicine applied in public services in France (Decree No. 82-453). This decree refers to the Labour Code, Public Health Code and Environmental Code.</p>
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                          <h3 id="SD-form" class="heavy">Formation received</h3>
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                            <p>To follow the legislation, we have been formed by Nathalie Doubrovine, the supervisor of the safety department of the LISBP. Her goal is to ensure both the well-being of the employees and the respect of safety rules and risk prevention. During the summer, we received different formations:
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                              <ul>
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                                <li><p>General information: two formations have been done, one during a meeting with Nathalie Doubrovine and the other with the software NEO. General information has been presented about the chemical, biological and fire risks. We also learned the prevention of those risks and the reaction to have when one of those risks occurs. At the end of the formation, a little test was done to see if everyone understood everything. Also, before using any new device, a quick formation is done.</p></li>
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                                <li><p>Autoclave formation: our team has attended an autoclave training, which showed us the explosive and implosive dangers of the dispositive and the security measures to take to protect ourselves. A lab coat, heat resistant gloves and glasses were required for the manipulation of the autoclave. The standard protocols of loading and unloading it were also demonstrated.</p></li>
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                                <li><p>Ethidium bromide room: because of the oncogenic risk of the ethidium bromide, we have a specific room where we must work with this solution. Nothing can get outside the room when it comes inside and everyone in the room must wear protection gloves, a lab coat and protection goggles. UV are also used in this room and protection mask must be wore during its use.</p></li>
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                              </ul>
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                            </p>
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                    </div>
 
                      
 
                      
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                    <ul>
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                            <h2 id="KR" class="heavy">Knowing the risks and minimize them</h2><hr/>
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                    <li> <p><i>Cloning of the part encoding Sirius</i></p></li>
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                    <li> <p><i>Overexpression and purification of Sirius</i></p></li>
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                    <li> <p><i>Fixation of Sirius to cellulose</i></p></li>
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                    </ul>
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                    </p>
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                    <h3 id="Materials_Methods_Sirius" class="heavy">Materials and Methods</h3>
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                    <p>
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The fused CBM3a and mRFP1 sequences were cloned into the pET28 expression vector in fusion with a histidine tag (Figure 1).
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</p>
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<figure class="figure"  style="text-align:center;">
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<img style="width : 70%; height: auto;" src="https://static.igem.org/mediawiki/2018/4/4f/T--Toulouse-INSA-UPS--Results--Youn--constructSirius.png" class="figure-img img-fluid rounded" alt="A generic square placeholder image with rounded corners in a figure.">
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<figcaption class="figure-caption"><strong>Figure 1:</strong> scheme of the Sirius construction</figcaption>
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</figure>
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<p>The resulting construct was transformed into <em>E. coli</em> strain and expression of the recombinant protein was induced using IPTG. The His-tagged protein was then purified on IMAC resin charged with cobalt and used in cellulose pull down assays. For the experimental details, see (link).</p>
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                    <h3 id="Results_Discussion_Sirius" class="heavy">Results and Discussion</h3>
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                    <h4 id="Production_purification_Sirius" class="heavy">Production and purification of Sirius</h4>
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<p>Interestingly, <i>E. coli</i> turned a deep pink shade after induction, indicating a strong production of Sirius (Figure 2).</p>
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<div style="text-align:center">
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<figure class="figure" id="figure" style="text-align:center;">
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<img style="width : 40%; heigth = auto;" src="https://static.igem.org/mediawiki/2018/9/96/T--Toulouse-INSA-UPS--Results--Youn--fig2sirius.jpg" class="figure-img img-fluid rounded" alt="SDS Page">
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<figcaption id="figcaption" class="figure-caption"><i><strong>Figure 2:</strong> E. coli expressing Sirius </i></figcaption>
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<div style="text-align:center">
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<figure class="figure" id="figure" style="text-align:center;">
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<img style="width : 60%; heigth = auto;" src="https://static.igem.org/mediawiki/2018/c/c3/T--Toulouse-INSA-UPS--Collaborations--GB--SDSPAGE_Analysis.jpg" class="figure-img img-fluid rounded" alt="SDS Page">
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<figcaption id="figcaption" class="figure-caption"><i><strong>Figure 3:</strong> SDS-PAGE analysis of Sirius purification. (CFE: cell free extract, FT: flow through, W: washes, E1/40: elution with 40mM imidazole, E1/100: elution with 100 mM imidazole, E2/100: elution with 100 mM imidazole, E1/300: elution with 300 mM imidazole, MW: molecular weight ladder.) </i></figcaption>
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</figure>
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<p>
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As shown in Figure 3, we successfully purified the Sirius protein. Indeed, induction with IPTG produced a large amount of a protein at the expected size for Sirius (52 kDa, lane CFE). This protein was then found predominant in elution samples (E1/40 and E1/100). We estimated the degree of purity of full-length Sirius at about 72%. In addition to the full-length protein, we observed several extra bands that likely correspond to proteolysis products.
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</p>
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                    <h4 id="Validation_Sirius" class="heavy">Validation</h4>
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                    <p>
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Once produced in <em<E. coli</Em>, Sirius binding to cellulose was tested using pull down assays. 70 &mu;M of Sirius protein or mRFP1 (without CBM3a) or buffer were incubated with cellulose. After five washes with resuspension buffer, cellulose pellets were recovered and the associated fluorescence was measured.</p>
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                    <p>
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When mRFP1 was added alone to cellulose, its associated colour mostly remained in the supernatant (Figure 4). When Sirius was associated to cellulose, the pinkish colour was clearly associated to the cellulose pellet. Quantification of the cellulose-associated fluorescence backed up these observations (Figure 5): control experiments showed that only background levels of fluorescence are retained in the cellulose pellet incubated with mRFP1 alone. These experiments have been performed in triplicate and statistical analyses indicate that the high level of fluorescence obtained with Sirius is definitively significant. These results clearly show that the CBM3a domain of Sirius interacts with cellulose, and thus mediates binding of mRFP1 protein to cellulose.
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                    </p>
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<div style="text-align:center">
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<figure id="figure2" class="figure"  style="text-align:center;">
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  <img style="width : 60%; heigth = auto;" src="https://static.igem.org/mediawiki/2018/6/61/T--Toulouse-INSA-UPS--Results--Youn--CPDSirius.png" class="figure-img img-fluid rounded" alt="Fluorescence Retained">
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<figcaption id="figcaption2" class="figure-caption"><i><strong>Figure 4:</strong> Picture of cellulose pull-down assay with mRFP1 alone (left tube) or with the Sirius construction (right tube).</i></figcaption>
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</figure>
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  </div>
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<div style="text-align:center">
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  <figure id="figure2" class="figure"  style="text-align:center;">
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    <img style="width : 60%; heigth = auto;" src="https://static.igem.org/mediawiki/2018/b/bb/T--Toulouse-INSA-UPS--Collaborations--GB--FluoRetained.jpg" class="figure-img img-fluid rounded" alt="Fluorescence Retained">
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      <figcaption id="figcaption2" class="figure-caption"><i><strong>Figure 5:</strong> Binding of Sirius to cellulose. Fluorescence retained in the cellulose pellet after pull down (triplicate test)</i></figcaption>
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  </figure>
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<div class="tab-pane fade" id="Results_Part2" role="tabpanel">
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<h2 id="Orthos" class="heavy">Orthos: Streptavidin and biotinylated compounds</h2>
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<hr/>
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<h3 id="Background_Orthos" class="heavy">Background</h3>
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                    <p>
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Once we proved the efficiency of the binding molecular platform of our system, CBM3a, we assessed the binding capacity of the first linker: the biotinylated molecule-binding module streptavidin.
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                    </p>
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<p>
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Orthos, named after the guardian of Geryon's cattle, is a fusion protein between CBM3a and a monomeric streptavidin head. The strong affinity of streptavidin for biotin (dissociation constant of 10<SUP>-13</SUP>M) will allow to tightly bind biotinylated organic molecules to Orthos.  We used  two types of biotinylated compounds to monitor the ability of our platform to functionalize cellulose:  fluorophores (mtag BFP and fluorescein) and  antimicrobial peptide (scygonadin).
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                    </p>
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                    <h3 id="Key_Achievements_Orthos" class="heavy">Key Achievements</h3>
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                    <p>
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                    <ul>
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                    <li> <p><i>Cloning of the part encoding Orthos</i></p></li>
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                    <li> <p><i>Production of Orthos carrying monomeric streptavidin</i></p></li>
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                    <li> <p><i>Production of biotinylated fluorophores</i> in vitro <i>and</i> in vivo</p></li>
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<li> <p><i>Functionalization of cellulose with biotinylated compounds</i></p></li>
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                    </ul>
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                    </p>
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                    <h3 id="Materials_Methods_Orthos" class="heavy">Materials and Methods</h3>
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                    <p>
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The fusion between monomeric streptavidin and CBM3a was cloned into the pET28 expression vector. The resulting construct was transformed into <i>E. coli</i> strain BL21 and expression of the recombinant protein was induced using IPTG. The protein was then purified on Regenerated Amorphous Cellulose (RAC) and used in cellulose pull down assays. For the experimental details, see <a href="https://2018.igem.org/Team:Toulouse-INSA-UPS/Experiments"> Experiments<a>.
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                    </p>
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                            <h3 id="KR-haz" class="heavy">Hazards we could met</h3>
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                    <h3 id="Results_Discussion_Orthos" class="heavy">Results and Discussion</h3>
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                    <h4 id="Purification_Orthos" class="heavy">Purification of Orthos</h4>
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                              <p>In the laboratory, there is a large variety of risks we could met. We will detail what risks could occurs and how the LISBP managed them here.</p>
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                              <h4>Main hazards:</h4>
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<div style="text-align:center">
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                              <p><u> External risks (earthquake, security issues…):</u></p>
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      <figure id="figure3" class="figure"  style="text-align:center;">
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                              <p>LISBP is concerned by many different types of hazards due to a wide spectrum of scientific activities. One hazard category gathers risks that are not directly linked to the LISBP research activity such as the risks due to working conditions (noise, thermal atmosphere, etc.), electrical hazards, work on computer screens, falls, etc. The main hazard categories concern the substances handled for research purposes such as class 1 microorganisms (GMO or not), urban wastewaters, chemical products among including CMRs, cryogenic fluids, etc… A second main hazard category relates to equipment and pilot plants with specific risks such as equipment using pressurized liquids, gases or steam (autoclaves), instruments generating non-ionizing radiations (Laser, UV lights) and electromagnetic radiations (RMN).</p>
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        <img style="width : 45%; heigth = auto;" src="https://static.igem.org/mediawiki/2018/d/d9/T--Toulouse-INSA-UPS--Collaborations--GB--SDSPAGE_Analysis2.jpg" class="figure-img img-fluid rounded" alt="SDS-PAGE">
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        <figcaption id="figcaption3" class="figure-caption"><i><strong>Figure 3:</strong> SDS-PAGE analysis of purification of Orthos (S: supernatant, W1, W2: washes, E: elution, MW: molecular weight ladder)</i></figcaption>
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    </figure>
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                              <h4>Hazardous chemicals:</h4>
−
    </div>
+
               
−
<p>
+
                              <p><u>List of hazardous, chemicals & inventory:</u></p>
−
Figure 3 presents the results of the purification of Orthos on RAC. The supernatant (S) contained all proteins produced in <i>E. coli</i>, and washes W1 and W2 allowed to eliminate most of non-specific interactions with cellulose. The elution step, using ethylene glycol, released Orthos protein from RAC. The band at about 40 kDa corresponds to Orthos (expected size: 39 kDa) and the band at 15 kDa likely corresponds to a proteolysis fragment of Orthos containing only the CBM3a module.
+
               
−
                    </p>
+
                              <p>The list of hazardous, chemicals and inventory is managed independently by the each LISBP sub group. A common LISBP list of CMR substances is monitored and updated.</p>
−
<p>
+
               
−
We estimated that the purification levels of the monomeric Orthos was about 45%, which is sufficient for our validation assays.
+
                              <p><u>Waste management plan:</u></p>
−
                    </p>
+
               
 +
                              <p>The chemical waste is managed in accordance with the environmental code. First, the waste is stored on a specially adapted LISBP/INSA site, before being disposed by a subcontracted service provider in accordance with the ADR rules concerning the waste treatment, valorization or destruction. Each waste removal is accompanied by tracking form for monitoring of hazardous waste.</p>
 +
               
 +
                              <p><u>Vacuum/pressurized equipment:</u></p>
 +
               
 +
                              <p>Pressurized equipment is monitored according to the Decree from 15 March 2000 concerning the work with the vacuum and high-pressure equipment.</p>
 +
               
 +
                              <p><u>Cryogenic/high temperature equipment:</u></p>
 +
               
 +
                              <p>Implementation of adequate means of prevention.</p>
 +
               
 +
                              <h4>Biological safety:</h4>
 +
               
 +
                              <p><u>Biological safety plan:</u></p>
 +
               
 +
                              <p>The laboratory applies the measures states in the articles of French Labour Code: L.4421-1 et R. 4421-1 a R. 4427-5 (Directive 90/619 on life insurance from 26 November 1990).</p>
 +
               
 +
                              <p><u>Biological material Type of organism used:</u></p>
 +
               
 +
                              <p>Prokaryotes, unicellular eukaryotes. Risk group: Risk group 1 Origin: E. coli and yeast mainly. Other biological: DNA, RNA... Facilities/Equipment Biosafety level: The majority of the LISBP facility is operated at Biosafety Level 1.</p>
 +
               
 +
                              <p><u>Biosafety cabinets:</u></p>
  
−
  <h4 id="Validation_Orthos" class="heavy">Validation of monomeric Orthos</h4>
+
                              <figure class="figure"  style="text-align:center;">
−
<h5 id="Validation_Invivo_Orthos" class="heavy">Validation using<i> in vivo </i>biotinylation</h5>
+
                                <img style="width : 70%; heigth = auto;" src="https://static.igem.org/mediawiki/2018/e/e9/T--Toulouse-INSA-UPS--Microbiology-Security-Hood.jpg" class="figure-img img-fluid rounded" alt="A generic square placeholder image with rounded corners in a figure.">
−
                    <p>
+
                                <figcaption class="figure-caption"><strong>Figure 1:</strong> Microbiology Safety Hood at our lab</figcaption>
−
In a first set of experiments, we assessed the ability of Orthos to functionalize cellulose with a compound biotinylated <i>in vivo</i>. For that purpose, we took advantage of a novel system for <i>in vivo</i> protein biotinylation that makes use of the biotin ligase BirA. Indeed, proteins containing the AviTag small peptide can be efficiently biotinylated <i>in vivo</i> by BirA. We thus co-expressed BirA and a version of the Blue Fluorescent protein (BFP) fused to the Avitag in <i>E. coli</i>. We added biotin in the culture medium during IPTG induction, in order to produce and purify biotinylated BFP.
+
                            </figure>
−
                    </p>
+
               
−
+
                              <p>The use of PSM type II guarantee the sterility of the manipulation and the protection of the environment and of the manipulator.</p>
−
<div style="text-align:center">
+
               
−
      <figure id="figure4" class="figure"  style="text-align:center;">
+
                              <p><u>Decontamination plan:</u></p>
−
        <img style="width : 50%; heigth = auto;" src="https://static.igem.org/mediawiki/2018/0/04/T--Toulouse-INSA-UPS--Collaborations--GB--FluoRetainedOrthos.jpg" class="figure-img img-fluid rounded" alt="Fluorescence Retained">
+
               
−
        <figcaption id="figcaption4" class="figure-caption"><i><strong>Figure 4:</strong> Functionalization of cellulose with Orthos bound to biotinylated BFP. Fluorescence remaining on cellulose fraction after several washes (*Mann Whitney test p-value 0.01)</i></figcaption>
+
                              <p>Certain laboratory rooms are subjected to gaseous decontamination once per year or upon request.</p>
−
    </figure>
+
               
−
    </div>
+
                              <p><u>Biological waste management plan:</u></p>
−
+
               
−
<p>
+
                              <p>The biological waste (liquid or solid) is pretreated chemically (bleach) or by autoclaving. Glass, sharp or jagged objects are disposed via a specialized company (DASRI).</p>
−
We then incubated 3.2 µM of Orthos with 32 µM of <i>in vivo</i> biotinylated BFP. For the control experiment BFP (without Orthos), the same quantity of BFP was added to have a relevant comparison. These samples were then incubated with cellulose. After three washes with resuspension buffer, fluorescence was measured in the cellulose pellets. As shown in Figure 4, fluorescence is twice more intense in Orthos sample than in the control sample containing BFP alone. These experiments have been performed in quadruplicate and a Mann Whitney statistical test indicated that the increased fluorescence signal obtained with Ortos is significant.
+
               
−
</p>
+
                              <p><u>Biological emergency plan (exposure spill):</u></p>
−
+
               
−
<p>
+
                              <p>In case of exposure spill, the liquids are absorbed through suitable means and further treated as biological waste.  </p>
−
These results demonstrate that Orthos protein binds efficiently to an <i>in vivo</i> biotinylated compound. In addition, they show that Orthos binds to cellulose and thus provide a proof of principle that Orthos design allows functionalization of cellulose with a fluorophore through its streptavidin linker.
+
               
−
</p>
+
                            <h3 id="KR-haz" class="heavy">Good laboratory practices</h3>
−
+
               
−
<h5 id="Validation_Invitro_Orthos" class="heavy">Validation using <i>in vitro</i> chemical biotinylation</h5>
+
                              <p>We have received a formation about the safety in the lab that teaches us some basic rules. Those rules are listed in the summary diagram below.</p>
−
                    <p>
+
                                 
−
In a second set of experiments, we tested the ability of Orthos to functionalize cellulose with <i>in vitro</i> biotinylated compounds. We used the newly emerging technique Cu(I)-free strain-promoted alkyne-azide click chemistry (SPAAC) allowing to couple <i>in vitro</i> a molecule containing a dibenzocyclooctyne (DBCO) moiety to another molecule bearing an azide function. We used this technique to ligate <i>in vitro</i> biotin-DBCO to an azide-functionalized fluorescein (FITC), thus leading to the expected biotinylated FITC.
+
                     </div>
−
                    </p>
+
                     <div class="tab-pane fade" id="Safe_Part3" role="tabpanel">
−
+
 
−
<div style="text-align:center">
+
                            <h2 id="SPC" class="heavy">Safety and project construction</h2><hr/>
−
      <figure id="figure5" class="figure"  style="text-align:center;">
+
−
        <img style="width : 45%; heigth = auto;" src="https://static.igem.org/mediawiki/2018/5/52/T--Toulouse-INSA-UPS--Collaborations--angeline--FluoRetainedOrthos2.jpg" class="figure-img img-fluid rounded" alt="Fluorescence Retained">
+
−
        <figcaption id="figcaption5" class="figure-caption"><i><strong>Figure 5:</strong> Functionalization of cellulose with Orthos bound to in vitro-biotinylated FITC. Fluorescence remaining on cellulose pellet fraction after several washes (quadruplicate test). *Mann Whitney test p-value 0.1</i></figcaption>
+
−
    </figure>
+
−
    </div>
+
−
+
−
<p>
+
−
Then, 58.0 µM of biotinylated FITC was incubated with 5.8 µM of Orthos protein. The same amount of FITC alone (58.0 µM) was used for the control experiment. These samples were incubated with cellulose and after five washes with resuspension buffer, fluorescence levels were measured in the cellulose pellet fractions (Figure 5). In the presence of Orthos, fluorescence in the cellulose pellet was about twice higher than in control experiments corresponding to the cellulose pellet incubated with FITC alone (without Orthos) or with the reaction buffer only. These experiments was performed performed in quadruplicate and a Mann Whitney statistical test indicated that the increased fluorescence signal obtained with Ortos is significant. This result clearly shows that purified Orthos retains the ability to mediate the interaction between cellulose and a compound biotinylated <i>in vitro</i>. In addition, this result provides another strong evidence that Orthos allows to functionalize cellulose using a fluorescent molecule through its streptavidin linker.
+
−
</p>
+
−
+
−
<h5 id="Validation_Scygonadin_Orthos" class="heavy">Validation using scygonadin</h5>
+
−
                    <p>
+
−
                    In order to confirm that the streptavidin moiety of Orthos is able to interact with different kind of biotinylated compounds, we decided to bind it to an antimicrobial peptide, scygonadin. To biotinylate this compounds, we used the in vivo protein biotinylation system as the one used for BFP molecule. We co-expressed BirA with a version of scygonadin fused to the Avitag in Pichia pastoris.                   </p>
+
−
<div style="text-align:center">
+
−
      <figure id="figure6" class="figure"  style="text-align:center;">
+
−
        <img style="width : 55%; heigth = auto;" src="https://static.igem.org/mediawiki/2018/5/55/T--Toulouse-INSA-UPS--Collaborations--angeline--Halo.jpg" class="figure-img img-fluid rounded" alt="Inhibition halo">
+
−
        <figcaption id="figcaption6" class="figure-caption"><i><strong>Figure 6:</strong> Halo of inhibition of Orthos alone ( C), scygonadin alone (S) and Orthos+scygonadin (C+S) samples</i></figcaption>
+
−
    </figure>
+
−
    </div>
+
−
<p>
+
−
<b>A compléter!</b>We then incubated Orthos with in vivo biotinylated scygonadin and we observed after incubation overnight halos of inhibition for each samples (Figure 6). We noticed an inhibition halo more important for the Orthos-scygonadin sample than the scygonadin alone control.  Unfortunately, we repeated this test, but we did not obtained the same result. Therefore, we need to improve our experiment to prove the coupling between Orthos and biotinylated scygonadin. These results are nevertheless encouraging in the perspective of functionalizing cellulose using Orthos bound to antibiotic protein.
+
−
</p>
+
−
+
−
<h3 id="Perspectives_Orthos" class="heavy">Perspectives</h3>
+
−
<p>
+
−
Our experiments using different kind of biotinylated compounds provide convincing proofs of concept of concept that Orthos can be conjugated, via its streptavidin head, to biotinylated organic molecules. The resulting molecules interact with cellulose and are therefore potent cellulose functionalizing compounds.
+
−
                    </p>
+
−
<p>
+
−
We can now envision to exploit the streptavidin linker of Orthos to ligate various compounds that can be biotinylated chemically (<i>in vitro</i>) or <i>in vivo</i>, <i>via BirA</i> co-expression.
+
−
                    </p>
+
−
<p>
+
−
In addition, we incorporated a TEV protease cleavage site between the N-terminus linker and the streptavidin. This allow to produce Orthos-bound biotinylated compounds that can be released by TEV cleavage if required, as in the case of odorant molecules for examples to a commercial use.
+
−
                    </p>
+
−
</div>
+
−
+
−
<!--PART 3 : cerberus-->
+
−
<div class="tab-pane fade" id="Results_Part3" role="tabpanel">
+
−
<h2 id="Cerberus" class="heavy">Cerberus: AzF</h2>
+
−
<hr/>
+
−
+
−
<h3 id="Background_Cerberus" class="heavy">Background</h3>
+
−
                    <p>
+
−
Cerberus, named after the guardian of the gates of the Underworld, is our three-headed protein made of the cellulose-binding molecular platform (CBM3a) fused at the N-terminus to the biotinylated molecule-binding head (monomeric streptavidin) and at the C-terminus to the unnatural amino acid azido-L-phenylalanine (AzF). The incorporation of AzF is achieved through the recognition of the amber stop codon at the C-terminus of CBM3a by an orthogonal AzF-charged tRNA.  
+
−
                    </p>
+
−
<p>
+
−
                    Coupling of molecules to the AzF head can be performed by click chemistry, either SPAAC or Cu(I)-catalyzed azide-alkyne click chemistry (CuAAC). To prove the versatility of the system, different compounds such as fluorescent proteins or paramagnetic beads have been clicked on Cerberus.
+
−
                    </p>
+
−
+
−
                    <h3 id="Key_Achievements_Cerberus" class="heavy">Key Achievements</h3>
+
−
                    <p>
+
−
                    <ul>
+
−
                    <li> <p><i>Cloning of the part encoding Cerberus</i></p></li>
+
−
                    <li> <p><i>Production of Cerberus</i></p></li>
+
−
                    <li> <p><i>Functionalization of cellulose</i></p></li>
+
−
                    </ul>
+
−
                    </p>
+
−
+
−
                    <h3 id="Materials_Methods_Cerberus" class="heavy">Materials and Methods</h3>
+
−
                    <p>
+
−
The fusion between the streptavidin linker and CBM3a platform sequences followed by the amber stop codon was cloned into the pET28 expression vector in fusion with the His-tag. <strong>The resulting construct was co-transformed into <i>E. coli</i> with pEVOL expression vector (coding for amino acyl tRNA synthetase (aarS)/tRNA orthogonal pair for <i>in vivo</i> AzF incorporation in <i>E. coli</i> under L-arabinose induction) into <i>E. coli</i> strain and expression of the recombinant protein was induced using IPTG and L-arabinose, with addition of AzF in the medium.</strong> The His-tagged protein was then purified on IMAC resin charged with cobalt and used in cellulose pull down assays. For the experimental details, see <a href="https://2018.igem.org/Team:Toulouse-INSA-UPS/Experiments"> Experiments<a>.
+
−
                    </p>
+
−
                    <h3 id="Results_Discussion_Cerberus" class="heavy">Results and Discussion</h3>
+
−
                    <h4 id="Purification_Cerberus" class="heavy">Production and purification of Cerberus</h4>
+
−
<p>
+
−
Cerberus production requires a deferred induction. We first induced the synthesis of aaRS/tRNA pair with L-arabinose, and added AzF at the same time. One hour later, we induced the production of Cerberus with IPTG.
+
−
                    </p>
+
−
+
−
                    <div style="text-align:center">
+
−
      <figure id="figure7" class="figure"  style="text-align:center;">
+
−
        <img style="width : 28%; heigth = auto;" src="https://static.igem.org/mediawiki/2018/7/70/T--Toulouse-INSA-UPS--Collaborations--angeline--SDSPAGE_cerberus.jpg" class="figure-img img-fluid rounded" alt="SDS-PAGE">
+
−
        <figcaption id="figcaption7" class="figure-caption"><i><strong>Figure 7:</strong>  SDS-PAGE analysis of the production of Cerberus with monomeric streptavidin (NI: non-induced, I-AzF: induced without AzF, I+AzF: induced with AzF, E1: elution with 40 mM imidazole, E2: elution with 100 mM imidazole, E3: elution with 100 mM imidazole, MW: molecular weight ladder)</i></figcaption>
+
−
    </figure>
+
−
    </div>
+
−
+
−
<p>
+
−
Cerberus production and purification were analysed on SDS gel (Figure 7). Induction in the presence of AzF (Figure 7, lane I+AzF) led to the expression of two major bands at 37 and 39 kDa approximately compared to control conditions, namely the non-induced (lane NI) and induced without AzF (lane I-AzF). We hypothesized that the upper band would correspond to Cerberus (expected molecular weight of monomeric streptavidin: 41 kDa), and the lower bands to Orthos proteins (Cerberus without the AzF and the His-tag). Surprisingly, both bands were present in the elution fractions which is incoherent since Orthos lacks an His-tag and was supposed to be washed out during purification steps. To confirm our hypothesis, we analysed the same fractions by western blot using antibodies detecting the His-tag (Figure 8).
+
−
                    </p>
+
−
+
−
<div style="text-align:center">
+
−
      <figure id="figure8" class="figure"  style="text-align:center;">
+
−
        <img style="width : 35%; heigth = auto;" src="https://static.igem.org/mediawiki/2018/0/01/T--Toulouse-INSA-UPS--Collaborations--angeline--SDSPAGE_cerberus2.jpg" class="figure-img img-fluid rounded" alt="Western blot">
+
−
        <figcaption id="figcaption8" class="figure-caption"<i><strong>Figure 8:</strong>  Western blot analysis of Cerberus production (NI: non-induced, I-AzF: induced without AzF, I+AzF: induced with AzF, E: elution, MW: molecular weight ladder)</i></figcaption>
+
−
    </figure>
+
−
    </div>
+
−
+
−
<p>
+
−
Anti-His-tag antibodies revealed  a band at about 45 kDa in the sample corresponding to IPTG induction in the presence of AzF (Figure 8, lane I+AzF). This band is not present in control samples (Figure 8, lane NI and I-AzF), indicating that it corresponds to Cerberus protein (theoretical size: 41 kDa). In addition to the full length protein , we observed several extra bands which very likely correspond to proteolysis products since they are detected with the anti-His-tag antibodies. Moreover, the band at 45 kDa is clearly detected in elution samples (Figure 8, lanes E). We estimated that the purification level of Cerberus with monomeric streptavidin in the elution samples was about 62%. These data show that Cerberus was efficiently purified and can be used for pull down assays. In addition, these results show that the experimental setup to produce Cerberus also leads to the production of Orthos when the amber stop codon is not recognized by the AzF-charged orthogonal  tRNA. Although Orthos does not contain a His-tag at its C-terminus, the protein seems to be efficiently co-purified with Cerberus. The basis of this observation is unclear but it may suggest that Orthos and Cerberus interact with each other, via their CBM3 or streptavidin moieties.
+
−
</p>
+
−
+
−
                     <h4 id="Validation_Cerberus" class="heavy">Validation</h4>
+
−
<h5 id="Validation_Invivo_Cerberus" class="heavy">Validation using FITC (Fluorescein isothiocyanate) molecules</h5>
+
−
                      
+
−
<div style="text-align:center">
+
−
      <figure id="figure7" class="figure"  style="text-align:center;">
+
−
        <img style="width : 50%; heigth = auto;" src="https://static.igem.org/mediawiki/2018/f/f8/T--Toulouse-INSA-UPS--Collaborations--angeline--FluoRetainedCerberus.jpg" class="figure-img img-fluid rounded" alt="Fluorescence Retained">
+
−
        <figcaption id="figcaption7" class="figure-caption"><i><strong>Figure 9:</strong> Fluorescence remaining in cellulose fraction after several washes (quadruplicate test). *Mann Whitney test p-value 0.03</i></figcaption>
+
−
    </figure>
+
−
    </div>
+
−
<p>
+
−
To test the potential of Cerberus in functionalizing cellulose, we monitored its ability to mediate an interaction between cellulose and a fluorescent compound. To generate a fluorescently labelled Cerberus protein, we performed a SPAAC reaction on 3.2 µM Cerberus protein using 31.9 µM µg of FITC-DBCO. In control experiment, 31.9 µM of FITC alone was used. These samples were then incubated with cellulose and after five washes with resuspension buffer, fluorescence levels were measured in the cellulose pellet fractions (Figure 9). We observed that the fluorescence levels in the cellulose pellet incubated with Cerberus protein clicked to FITC were about 3 times higher than in the control experiments corresponding to the cellulose pellet incubated with FITC alone. These results show that Cerberus can efficiently be conjugated to fluorescent molecules bearing a DBCO group by click chemistry and that the resulting fluorescent molecules strongly interact with cellulose. Therefore, Cerberus is both a convenient and potent platform to functionalize cellulose.
+
−
                    </p>
+
−
+
−
+
−
<h5 id="Validation_Invitro_Cerberus" class="heavy">Validation using paramagnetic beads</h5>
+
−
                    <p>
+
−
We further characterize the functionality of Cerberus in a second set of experiments  using paramagnetic beads. To bind paramagnetic beads to Cerberus, we performed a click reaction using 3.2 µM of Cerberus protein and 32 µM DBCO-conjugated paramagnetic beads. As a control experiment, 32 µM of paramagnetic beads were used. These samples were then incubated with cellulose, and after five washes with resuspension buffer, we measured the magnetisation of cellulose using a magnet.
+
−
                    </p>
+
−
<div style="text-align:center">
+
−
      <figure id="video1" class="figure"  style="text-align:center;">
+
−
        <video controls preload="auto" muted="true" style="width : 50%; heigth = auto;" src="https://static.igem.org/mediawiki/2018/1/14/T--Toulouse-INSA-UPS--Collaborations--angeline--ferocell.MOV" alt="Magnetic cellulose">
+
−
       
+
−
</video>
+
−
    <figcaption id="vidcaption" class="figure-caption"><i>Left: control with paramagnetic beads alone (no Cerberus); Right: Cerberus-paramagnetic beads</i></figcaption>
+
  
−
    </figure>
+
                            <p>Safety management, french legislation and ethics took an important part in the way we managed our project. The brainstorming have been particularly affected by those points. Indeed, we wanted to work on a project that can live outside the iGEM competition and outside the lab. So, very soon, we select our ideas depending on “will the use of GMOs become an issue for the safety of the customer by also for the environment”. Our project Cerberus need GMOs but we develop it to avoid any contact with the user of the product but also with the environnement. At the end, we produce a protein in laboratory with GMOs but only the protein, and not the organism that produced it, is used.</p>
−
    </div>
+
                            <p>If this part of the construction of our project interest you, we invite you to read the <a href="https://2018.igem.org/Team:Toulouse-INSA-UPS/Human_Practices">Human Practices section</a>.</p>
−
<p>
+
                               
−
                    As observed on the video, the cellulose incubated with the Cerberus protein conjugated to paramagnetic beads is quickly and totally attracted by the magnet. In contrast, in the control experiment, we only observed a slight movement of a part of the cellulose towards the magnet. This can be explained by the fact that washes do not remove all paramagnetic beads non-attached to the cellulose, or that paramagnetic beads naturally bind to cellulose but this interaction is not strong enough to resist washes.
+
                     </div>
−
</p>
+
                </div>
−
+
            </div>
−
<p>
+
−
These results show that Cerberus has the ability to interact simultaneously with cellulose and molecules with DBCO group, indicating that this modified protein, allows to functionalize cellulose through its linker containing unnatural amino acid (AzF).
+
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<h4 id="Validation_Scygonadin_Cerberus" class="heavy">Compounds functionalization</h4>
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To go further in our project, we decided to functionalyse two inorganic molecules, graphene and carbon nanotubes, using a reaction of diazotization. This experiment was performed with the help of the ENSIACET laboratory in Toulouse. To check functionalization, we analysed samples by Thermogravimetric Analysis (TGA). The concept of TGA consists in measuring the mass change of a sample depending on temperature and time, in a controlled atmosphere. We choose to perform the experiment under azote, an inert gas that avoids mass loss by oxidation, and we studied the mass change from 25 to 1,000 °C (15 °C/min).  
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        <img style="width:100%; heigth = auto;" src="https://static.igem.org/mediawiki/2018/e/ed/T--Toulouse-INSA-UPS--Collaborations--angeline--massloss1.jpg" class="figure-img img-fluid rounded" alt="Mass loss">
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        <figcaption id="figcaption10" class="figure-caption"><i><strong>Figure 10:</strong> Analysis of mass loss for functionalized CNT sample</i></figcaption>
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        <img style="width : 100%; heigth = auto;" src="https://static.igem.org/mediawiki/2018/c/cb/T--Toulouse-INSA-UPS--Collaborations--angeline--massloss2.jpg" class="figure-img img-fluid rounded" alt="Mass loss">
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        <figcaption id="figcaption11" class="figure-caption"><i><strong>Figure 11:</strong> Analysis of mass loss for non-functionalized CNT sample</i></figcaption>
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                    Figures 10 and 11 present the results of mass loss for functionalized Carbon Nanotube (CNT) and non-functionalized CNT samples, respectively. For functionalized CNT sample (Figure 10), we observed a mass loss of 27.4% around 100 °C corresponding to water loss. A second step of mass loss is observed around 550 °C, with a diminution of 19.8%. For the non-functionalized sample (Figure 11), we observed two mass losses at 150 and 750 °C corresponding to 1.5% and 3% of mass loss respectively. The significative difference between these two samples allowed us to conclude that the CNT functionalization has been successful.
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        <figcaption id="figcaption12" class="figure-caption"><i><strong>Figure 12:</strong> Analysis of mass loss for functionalized graphene sample</i></figcaption>
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        <img style="width : 100%; heigth = auto;" src="https://static.igem.org/mediawiki/2018/4/40/T--Toulouse-INSA-UPS--Collaborations--angeline--massloss4.jpg" class="figure-img img-fluid rounded" alt="Mass loss">
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        <figcaption id="figcaption13" class="figure-caption"><i><strong>Figure 13:</strong> Analysis of mass loss for non-functionalized graphene sample</i></figcaption>
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Figures 12 and 13 present the results of mass loss for functionalized graphene and non-functionalized graphene samples respectively. For functionalized graphene sample (Figure 12), we observed a mass loss of 31.0% around 100 °C corresponding to water loss. A second step of mass loss is observed around 500 °C, with a diminution of 19.0%. For the non-functionalized sample (Figure 13), we did not observe a significative mass loss. So we can conclude that the graphene functionalization has been successful.
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<h3 id="Perspectives_Cerberus" class="heavy">Perspectives</h3>
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Our experiments provide robust proofs of concept that Cerberus can be conjugated, via its unnatural amino acid AzF, to organic or inorganic molecules bearing a DBCO group by click chemistry. The resulting molecules strongly interact with cellulose and are therefore potent cellulose functionalizing compounds.
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We can now consider fixation of various compounds that can be chemically functionalized. We can also imagine double fixations, for example a fluorophore on the streptavidin head and paramagnetic beads on the AzF linker. This double fixation allows to envision endless possibilities to functionalize cellulose.
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Some microorganisms naturally produce cellulose. One of these is <i>Gluconacetobacter hansenii</i>, a species of acetic acid bacteria, which can produce cellulose as a biofilm to protect itself. We chose to work with this bacterium to produce cellulose, because the iGEM Imperial team 2014 has set up culture conditions to optimize cellulose production. We used the best culture conditions to obtain a maximum of cellulose.
 
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                    <li> <p><i>Production of bacterial cellulose</i></p></li>
 
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<i>G. hansenii</i> was grown in Hestrin-Shramm (HS) medium at 30 °C. After one week of culture, bacterial cellulose was extracted and purified.  For the experimental details, see <a href="https://2018.igem.org/Team:Toulouse-INSA-UPS/Experiments"> Experiments<a>.
 
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For the functionalization, bacterial cellulose was incubated with purified Sirius or with purified mRFP1 alone as a control. Cellulose was then washed several times with resuspension buffer to remove non-attached proteins.
 
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        <figcaption id="figcaption14" class="figure-caption"><i><strong>Figure 14:</strong> Picture of functionalized cellulose with Sirius (CBM3a fused with mRFP1). Left: control mRFP1 alone; Right: Sirius</i></figcaption>
 
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Figure 14 presents the results of the functionalization of our bacterial cellulose with a fluorescent protein. Only the cellulose sample incubated with Sirius (CBM3a-mRFP1) remains fluorescent after several washes with resuspension buffer. The control cellulose samples showed that only a weak coloration is retained after incubation with mRFP1 alone (not coupled with CBM3a platform). These results show that CBM3a interacts with the bacterial cellulose,  and allows fixation of the mRFP1 protein to cellulose.
 
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Now that we proved the capacity of CBM3a to interact with bacterial cellulose, we are confident that Orthos and Cerberus proteins should also interact with bacterial cellulose. Moreover, since we proved that we can conjugate several organic and inorganic compounds to the binding modules of Cerberus (streptavidin and AzF), we can envision functionalizing bacterial cellulose with many different compounds to provide it with new features.
 
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Revision as of 22:52, 12 October 2018

SAFETY

There is a lot of dangers in a laboratory. So, everything cannot be done and there are rules to respect to protect the environment and ourselves. Due to an atmosphere of mistrust concerning GMOs in France, it is also very important to us to appease the opinion with a strict safety policy.

First, we will present here how the safety department in INSA is organized: what kind of structure received us, what is the French legislation concerning risks and how we have been formed. Then, we will detail what kind of risks we could met during the lab work and how we should avoid them with good laboratory habits. Finely, we will explain how we constructed our project to minimize the health and safety issues.

INSA Safety Department

A generic square placeholder image with rounded corners in a figure.
Figure 1: Ethidium Bromide Handling Room.

Organization of the INSA Safety Department

We have been received by the LISBP. The head of the laboratory where we worked is Carole Jouve and the organization of the safety department is described in the figure 1 below. We are very grateful for the welcoming of our colleagues and the time they spend to form us.

organization chart
Figure 2: Organization chart of the INSA safety department.

Legislation and French labor Law

We are working in a french engineering school (INSA Toulouse), thus we have to respect the french national regulations about working conditions and manipulation of GMOs.

As we work with microorganisms, we are concerned by the regulation on workers protection against risks resulting from their exposure to pathogenic biological agents (Decree No. 94-352 of 4 May 1994). It also includes human endoparasites which may cause infections, allergies or toxicity. This Decree is the French transposition of the Directive 90/679 / EEC and is also transcribed in the Labour Code (Articles L4421-1 R4421-1 to R4427-5). This Decree of the 16th July 2007 describes the technical preventive measures that must be followed in research laboratories, where workers are likely to be exposed to biological pathogens. We must obey to the rules of health, safety, and preventive medicine applied in public services in France (Decree No. 82-453). This decree refers to the Labour Code, Public Health Code and Environmental Code.

Formation received

To follow the legislation, we have been formed by Nathalie Doubrovine, the supervisor of the safety department of the LISBP. Her goal is to ensure both the well-being of the employees and the respect of safety rules and risk prevention. During the summer, we received different formations:

  • General information: two formations have been done, one during a meeting with Nathalie Doubrovine and the other with the software NEO. General information has been presented about the chemical, biological and fire risks. We also learned the prevention of those risks and the reaction to have when one of those risks occurs. At the end of the formation, a little test was done to see if everyone understood everything. Also, before using any new device, a quick formation is done.

  • Autoclave formation: our team has attended an autoclave training, which showed us the explosive and implosive dangers of the dispositive and the security measures to take to protect ourselves. A lab coat, heat resistant gloves and glasses were required for the manipulation of the autoclave. The standard protocols of loading and unloading it were also demonstrated.

  • Ethidium bromide room: because of the oncogenic risk of the ethidium bromide, we have a specific room where we must work with this solution. Nothing can get outside the room when it comes inside and everyone in the room must wear protection gloves, a lab coat and protection goggles. UV are also used in this room and protection mask must be wore during its use.

Knowing the risks and minimize them

Hazards we could met

In the laboratory, there is a large variety of risks we could met. We will detail what risks could occurs and how the LISBP managed them here.

Main hazards:

External risks (earthquake, security issues…):

LISBP is concerned by many different types of hazards due to a wide spectrum of scientific activities. One hazard category gathers risks that are not directly linked to the LISBP research activity such as the risks due to working conditions (noise, thermal atmosphere, etc.), electrical hazards, work on computer screens, falls, etc. The main hazard categories concern the substances handled for research purposes such as class 1 microorganisms (GMO or not), urban wastewaters, chemical products among including CMRs, cryogenic fluids, etc… A second main hazard category relates to equipment and pilot plants with specific risks such as equipment using pressurized liquids, gases or steam (autoclaves), instruments generating non-ionizing radiations (Laser, UV lights) and electromagnetic radiations (RMN).

Hazardous chemicals:

List of hazardous, chemicals & inventory:

The list of hazardous, chemicals and inventory is managed independently by the each LISBP sub group. A common LISBP list of CMR substances is monitored and updated.

Waste management plan:

The chemical waste is managed in accordance with the environmental code. First, the waste is stored on a specially adapted LISBP/INSA site, before being disposed by a subcontracted service provider in accordance with the ADR rules concerning the waste treatment, valorization or destruction. Each waste removal is accompanied by tracking form for monitoring of hazardous waste.

Vacuum/pressurized equipment:

Pressurized equipment is monitored according to the Decree from 15 March 2000 concerning the work with the vacuum and high-pressure equipment.

Cryogenic/high temperature equipment:

Implementation of adequate means of prevention.

Biological safety:

Biological safety plan:

The laboratory applies the measures states in the articles of French Labour Code: L.4421-1 et R. 4421-1 a R. 4427-5 (Directive 90/619 on life insurance from 26 November 1990).

Biological material Type of organism used:

Prokaryotes, unicellular eukaryotes. Risk group: Risk group 1 Origin: E. coli and yeast mainly. Other biological: DNA, RNA... Facilities/Equipment Biosafety level: The majority of the LISBP facility is operated at Biosafety Level 1.

Biosafety cabinets:

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Figure 1: Microbiology Safety Hood at our lab

The use of PSM type II guarantee the sterility of the manipulation and the protection of the environment and of the manipulator.

Decontamination plan:

Certain laboratory rooms are subjected to gaseous decontamination once per year or upon request.

Biological waste management plan:

The biological waste (liquid or solid) is pretreated chemically (bleach) or by autoclaving. Glass, sharp or jagged objects are disposed via a specialized company (DASRI).

Biological emergency plan (exposure spill):

In case of exposure spill, the liquids are absorbed through suitable means and further treated as biological waste.

Good laboratory practices

We have received a formation about the safety in the lab that teaches us some basic rules. Those rules are listed in the summary diagram below.

Safety and project construction

Safety management, french legislation and ethics took an important part in the way we managed our project. The brainstorming have been particularly affected by those points. Indeed, we wanted to work on a project that can live outside the iGEM competition and outside the lab. So, very soon, we select our ideas depending on “will the use of GMOs become an issue for the safety of the customer by also for the environment”. Our project Cerberus need GMOs but we develop it to avoid any contact with the user of the product but also with the environnement. At the end, we produce a protein in laboratory with GMOs but only the protein, and not the organism that produced it, is used.

If this part of the construction of our project interest you, we invite you to read the Human Practices section.

No dogs were harmed over the course of this iGEM project.

INSA Toulouse UPS LISBP TWB Defi Adisseo Foundation ESF Catalyses


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