Difference between revisions of "Team:Pasteur Paris/Demonstrate"

 
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                 <p><a href="#Reconnect" class="link">Reconnect Nerves</a></p>
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                 <p><a href="#Nerves" class="link">Nerve Growth</a></p>
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                <p><a href="#Fight" class="link">Fight Infections</a></p>
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                 <p><a href="#Kill" class="link">Kill Switch</a></p>
 
                 <p><a href="#Kill" class="link">Kill Switch</a></p>
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                <p><a href="#Membrane" class="link">Membrane</a></p>
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                <p><a href="#Design" class="link">Design</a></p>
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                <p><a href="#Entrepreneurship" class="link">Entrepreneurship</a></p>
 
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             <div class="block title" id="Reconnect">
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             <div class="block title" id="Nerves">
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                 <h1>RECONNECT NERVES</h1>
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                 <h1>NERVE GROWTH FACTOR AND NEURONAL CULTURE</h1></div>
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                <i style="text-align: left;"><p>Achievements:<br>
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                    <ul>
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                        <li>Successfully cloned a part coding for secretion of NGF in pET43.1a and iGEM plasmid backbone pSB1C3, creating a new composite part <a href="http://parts.igem.org/Part:BBa_K2616000"> Bba_K2616000 </a> </li>
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                        <li>Successfully sequenced <a href="http://parts.igem.org/Part:BBa_K2616000"> Bba_K2616000 </a> in pSB1C3 and sent to iGEM registry </li>
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                        <li>Successfully co-transform E. coli with plasmid secreting NGF and plasmid expressing the secretion system, creating bacteria <b>capable of secreting NGF</b> in the medium</li>
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                        <li>Successfully characterized production of NGF thanks to mass spectrometry</li>
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                        <li>Successfully <b>observe axon growth</b> in microfluidic chip in presence of commercial NGF</li>
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                    </ul><br></p>
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                    <p>Next steps:<br>
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                    <ul>
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                        <li><b>Purify</b> secreted NGF, and characterize its effects on neuron growth thanks to our microfluidic device </li>
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                        <li><b>Global proof of concept</b> in a microfluidic device containing neurons in one of the chamber, and our engineered bacteria in the other</li>
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                    </ul>
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                </p></i>
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                <h4 style="text-align: left;">DNA assembly</h4><br><br>
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                 <p> We successfully designed and cloned a biobrick coding for the secretion of rat proNGF in pET43.1a and iGEM plasmid backbone pSB1C3, creating the new part <a href="http://parts.igem.org/Part:BBa_K2616000"style="font-weight: bold ; color:#85196a;"target="_blank"> Bba_K2616000 </a>. We <b>confirmed this genetic construct</b> by sequencing. </p>  
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                 <p>The <b>sequence</b> we designed codes for two different proteins: <b>proNGF (Nerve Growth Factor)</b> and <b>TEV protease</b> (from Tobacco Etch Virus). These two proteins are fused in C-terminal with a signal peptide for <i>E. coli</i> Type I Secretion System which consists in the last 60 amino-acids of HaemolysinA (<b>HlyA</b>). Each coding sequence is separated from the signal peptide by the cleavage sequence for TEV, in order to get the protein without its signal peptide (Figure 3).</p>
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<br>
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            </div>
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                 <p> We confirmed the production of proNGF in three different ways:
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            <div class="block half">
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                <ul style="text-align: left;">
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                <img src="https://static.igem.org/mediawiki/2018/8/80/T--Pasteur_Paris--ProNGFandTEVproduction.png">
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                <li>SDS PAGE followed by western blot.</li>
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                <div class="legend"><b>Figure 1: </b>proNGF and TEV production cassette </div>
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                <li>Mass spectrometry.</li>
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            </div>
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                <li>Activity on the growth of rat E18 cortical cells.</li>
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            <div class="block full">
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                </ul>
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                 <p>This DNA construct was ordered in two parts, named Seq1 (1096 bp) and Seq2 (1153 bp) in commercial plasmids pEX-A258 from gene synthesis. Seq1 and Seq2 were amplified in competent <i>E. coli</i> DH5-<FONT face="Raleway">α</FONT>.   After bacteria culture and plasmid DNA extraction, we digested commercial vectors with restriction enzymes (<b>NheI</b> and <b>BamHI</b> for Seq1, <b>MscI</b> and <b>HindIII</b> for Seq2). We extracted the inserts from the gel and performed a ligation by using specific overlaps into <b>linearized pET43.1a</b> for proNGF expression and into <b>pSB1C3</b> for iGEM sample submission.<br>We repeated the procedure (transformation in <i>E. coli</i>  Stellar competent cells, bacteria culture, plasmid DNA extraction, digestion) and we proved that our vector pet43.1a contained Seq1 and Seq2 (Figure 2) and that pSB1C3 contained Seq1 and Seq2 (Figure 3) after digestion and DNA electrophoresis. Plasmid DNA of pSB1C3 construction was purified and sent for sequencing (Figure 4).</p>
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<br>
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            </div>
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                <p> We designed and manufactured <b>microfluidic chips</b> in order to test our final proof of concept. </p>  
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            <div class="block half">
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                <p> We grew embryonic E18 rat neurons in our self-made microfluidic chips and successfully <b>observed axon growth</b> in the presence of commercial NGF and our recombinant proNGF. </p>
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                <img src="https://static.igem.org/mediawiki/2018/7/7c/T--Pasteur_Paris--gelNGFpET.png">
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                <p> To conclude, we showed that the activity of our recombinant proNGF was comparable to the one of commercial NGF used at concentrations of 500 to 900 ng/mL,  <b> demonstrating </b> that our theoretical idea works <i>in vitro</i>.</p>
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                <div class="legend"><b>Figure 2: </b> Agarose 1% gel after electrophoresis of digested pET43.1 containing Seq1 and Seq2 (Bba_K2616000) with NdeI. Colonies 6, 9, 10 ,11, 15 have the correct construction.</div>
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</div>
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            <div class="block half">
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                <img src="https://static.igem.org/mediawiki/2018/e/ef/T--Pasteur_Paris--gelNGFpSB1C3.png">
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                <div class="legend"><b>Figure 3: </b> Agarose 1% gel after electrophoresis of digested pSB1C3 containing Seq1 and Seq2 (Bba_K2616000) with EcoRI/PstI. Colonies 3, 7 and 8 have the correct construction.</div>
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             <div class="block title" id="Kill">
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                <p>Alignment of <b>Sequencing</b> Results then confirmed that pSB1C3 contained Seq1 and Seq2, <a href="http://parts.igem.org/Part:BBa_K2616000"> Bba_K2616000 </a>. </p>
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                 <h1>KILL SWITCH</h1></div>
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            </div>
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            <div class="block two-third center">
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                 <img src="https://static.igem.org/mediawiki/2018/e/ee/T--Pasteur_Paris--Sequencing_proNGF.PNG">
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                <div class="legend"><b>Figure 4: </b> Alignment of sequencing results for BBa_K2616000. Sequencing perform in pSB1C3 and three primers were designed (FOR1, FOR2, FOR3) to cover the whole sequence. Image from Geneious. </div>
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            </div>
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            <div class="block full">
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                <p>The construction was successfully assembled. On Figure 4, mismatches are visible which correspond to the reduced precision of sequencing after 600 bp. To avoid this lack of precision, we used three different primers, allowing us to cover the whole sequence without mistakes. As visible, the mismatches are only present at the extremities of each primer sequencing. </p>
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            </div>
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          <div class="block two-third center">
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                 <h4 style="text-align: left;">proNGF characterization and purification</h4><br><br>
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                 <p> We successfully designed and cloned a biobrick coding for a temperature sensitive kill-switch, <b>creating the new part</b> <a href="http://parts.igem.org/Part:BBa_K2616002"style="font-weight: bold ; color:#85196a;"target="_blank"> Bba_K2616002 </a>. We <b>confirmed this genetic construct</b> by sequencing. </p>
 +
<br>
 +
                        <p>We observed the survival and normal growth of our engineered chassis <i>E. coli</i> BL21(DE3)pLysS at 25°C and 37°C and the <b>absence of growth</b> at 18°C and 20°C. Comparatively, our negative control, the same chassis, transformed with an empty vector, grew normally at all temperatures tested.</p>
 +
<br>
 +
                        <p>To conclude, we have <b>demonstrated</b> that our kill-switch is a very efficient way to prevent the contamination of the environment by our genetically modified bacteria in case of accidental release.</p>  
  
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    <p> Our chassis is <b><i>Escherichia coli </i>BL21(DE3) pLysS</b>, a specific strain dedicated to producing high amounts of desired proteins under a T7 promoter. Thus, we co-transformed our bacteria with <a href="http://parts.igem.org/Part:BBa_K2616000"> Bba_K2616000 </a> and pVDL 9.3, generously provided by Dr. Victor de Lorenzo, from Centro Nacional de Biotecnologia of Madrid, bearing HlyB and HlyD (Type I secretion system) sequences, in order to get a chance to secrete NGF out of the cell.<br><br>
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            </div>
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Bacteria were grown at a large scale (800 mL), and proNGF expression was induced with 0.1 mM IPTG for 2 hours at 37°C. <br><br>
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We tried to achieve His-tagged proNGF purification using Ni-NTA affinity purification column. We eluted our protein using a gradient of imidazole-containing buffer and one peak was detected.<br><br></p>
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            </div>
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            <div class="block two-third center">
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                <img src="https://static.igem.org/mediawiki/2018/6/69/T--Pasteur_Paris--ResultsFPLC.png">
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                <div class="legend"><b>Figure 5: </b>FPLC proNGF purification with ÄKTA pure (General Electric) Ni-NTA column was equilibrated with buffer A (50 mM Tris, pH 7.4, 200 mM NaCl). Supernatant of lyzed bacteria was introduced through the column. Washing with 5% of buffer B. Elution by buffer B gradient (buffer A + imidazole 250 mM). UV absorbance at 280nm is shown in blue, conductivity in red, and concentration of buffer B in green. </div>
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            </div>
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            <div class="block full">
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                <p>We analyzed bacterial lysate and purification fractions using SDS-PAGE electrophoresis and Mass spectrometry. </p>
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            </div>
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            <div class="block half">
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                <img src="https://static.igem.org/mediawiki/2018/5/53/T--Pasteur_Paris--SDSPage.png">
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                <div class="legend"><b>Figure 6: </b>SDS-PAGE gel Bis-Tris 4-12% of bacterial lysate and proNGF purification fraction by SDS-PAGE.
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</div>
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            </div>
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            <div class="block half">
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<p> The proNGF purification using NiNTA column is not conclusive. Many proteins are found on elution fractions. His-tagged proNGF fused to HlyA export signal should be found at 33 kDa while the proNGF cleaved by TEV protease should be found at 27 kDa. We finally analyzed five gel bands of the FPLC flow-through (lane 2, Figure 6) by mass spectrometry, by LC/MS/MS, to verify the presence of proNGF.</p>
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</div>
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<div class="block half">
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<p>According to Figure 7, proNGF pattern are found on each lane sent to mass spectrometry. The major amount is found on fraction 5, corresponding to 33 kDa, at this molecular weight, the proNGF is still fused to the signal export. The TEV protease, 34 kDa fused to signal export and 28 kDa cleaved from the signal export are found.  </p>
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</div>
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            <div class="block half">
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                <img src="https://static.igem.org/mediawiki/2018/c/cd/T--Pasteur_Paris--distribution.png">
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                <div class="legend"><b>Figure 7: </b>Distribution of proNGF and TEV protease by gel fractions after mass spectrometry analysis. </div>
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            </div>
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            <div class="block full">
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<p>Analysis of Fraction 5 of the gel shows our protein proNGF is present but is still bound to its signal peptide HlyA. (Figure 8) Mass spectrometry spectrum of Peptide A, IDTACVCVLSR, from proNGF sequence is shown in Figure 9. Mass spectrometry spectrum of Peptide B, IISAAGSFDVKEER from fused HlyA signal export is shown in Figure 9. The presence of mass spectrometry identified peptides corresponding to the fusion of proNGF and HlyA indicate some proNGF uncleaved from the signal export</p>
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</div>
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            <div class="block two-third center">
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                <img src="https://static.igem.org/mediawiki/2018/b/ba/T--Pasteur_Paris--Align_sequence_Mass.png">
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                <div class="legend"><b>Figure 8: </b>Alignment sequences of proNGF fused to HlyA export signal and peptides identified by mass spectrometry. In light blue peptides that match proNGF amino acids sequence. In light yellow, peptides that match HlyA signal export. Sequence has been annotated to match corresponding protein amino acid sequences : In orange His tagged proNGF, in red TEV protease cleaving site, in rose HlyA signal export.</div>
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            </div>
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            <div class="block two-third center">
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                <img src="https://static.igem.org/mediawiki/2018/2/20/T--Pasteur_Paris--massspec.png">
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                <div class="legend"><b>Figure 9: </b>Mass spectrometry spectrum. A) Peptide identified corresponding to proNGF. B) Peptide identified corresponding to the fusion of proNGF and HlyA export signal. </div>
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            <div class="block full">
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                <p>The proNGF did not seem to be retained on the affinity column. We performed batch purification using NiNTA beads under native and partial denaturing conditions (Urea 2 M) followed by Western Blot analysis with immunodetection through Anti-His Antibodies Alexa Fluor 647. (Figure 10) Detection of His-tag in the pellet supernatant of induced BL21 with 1 mM IPTG and flow through when partially denatured.</p>
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<p> His-tagged proNGF was not retained on NiNTA beads. N-terminal His tag may be hidden in the protein fold. Consequently, we did not manage to purify the proNGF.
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</p></div>
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            <div class="block two-third center">
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                <img src="https://static.igem.org/mediawiki/2018/5/56/T--Pasteur_Paris--WBproNGF.png">
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                <div class="legend"><b>Figure 10: </b>Western Blot analysis of batch purification proNGF under native and partial denaturing conditions. </div>
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             <div class="block title" id="Fight">
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             <div class="block title" id="Membrane">
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                 <h1>FIGHT INFECTIONS</h1><br>
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                 <h1>MEMBRANE BIOCOMPATIBILITY AND CONDUCTIVITY</h1>
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                <i style="text-align: left;"><p>Achievements:<br>
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        </div>
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                    <ul>
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          <div class="block two-third center">
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                        <li>Successfully cloned a part coding for RIP secretion in pBR322 and in pSB1C3, creating a new part <a href="http://parts.igem.org/Part:BBa_K2616001"> Bba_K2616001 </a>.
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              <p> In search of a <b>biocompatible conductive polymer</b> to confine bacteria, we successfully polymerized <b>PEDOT:Ts</b> and <b>PEDOT:Cl</b> on <b>alumina oxyde membrane filters</b>. We also partially polymerized <b>PEDOT:PSS</b>.</p>
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                        <li>Successfully sequenced <a href="http://parts.igem.org/Part:BBa_K2616001"> Bba_K2616001 </a> in pSB1C3 and sent to iGEM registry.
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<br>
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                        <li>Successfully cultivated S. aureus biofilms in 96 well plates with different supernatants.</li>
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              <p> We <b>demonstrated</b> that a polymer-coating <b>enhances the electrical properties</b> of the membranes as PEDOT:Ts-coated and PEDOT:Cl-coated membranes are <b>more conductive</b> than uncoated membranes. Moreover, experiments showed a <b>better biocompatibility</b> for the polymer-coated membranes compared to the gold-coated ones. </p>
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                    </ul><br></p>
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<br>
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                    <p>Next steps:<br>
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                 <p> To conclude, we successfully <b>demonstrated</b> that our membrane could confine bacteria. This feature is one of the essential safety components that we have engineered in our NeuronArch project, ensuring the patient's safety.</p>
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                    <ul>
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                        <li>Clone the sensor device with inducible RIP production upon S. aureus detection.</li>
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                        <li>Improve the characterization of RIP effect on biofilm formation.</li>
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                    </ul>
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                </p></i>
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            </div>
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            <h2>RIP Secretion <a href="http://parts.igem.org/Part:BBa_K2616001"> BBa_K2616001</a></h2><br><br>
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                <p>The <b>sequence</b> we designed contains two <b>RIP (RNAIII Inhibiting Peptide)</b> sequences fused to two different export signal peptides for <i>E. coli</i> Type II Secretion System: <b>DsbA</b> and <b>MalE</b>, placed on N-terminal. (image: Figure 1. Schematic representation of the RIP production cassette. The cassette is composed of RIP sequence (blue) fused to DsbA signal (green) and further RIP sequence again (green) fused to MalE signal (red).)<br><br></p>
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            <div class="block two-third center">
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          <img src="https://static.igem.org/mediawiki/2018/f/fd/T--Pasteur_Paris--BBa_K2616001.png">
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          <div class="legend"><b>Figure 11: </b>proNGF and TEV production cassette </div>
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              </div>
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                 <p>Once we received the sequence encoding for this production cassette named Seq8 (461bp) in commercial plasmid pEX-A258 by gene synthesis. Plasmids was amplified in competent <i>E. coli</i> DH5alpha. After bacteria culture and plasmid DNA extraction, we digested commercial vector with <b>EcoRI</b> and <b>PstI</b> restriction enzymes. We extracted the inserts from the gel and performed a ligation by using specific overlaps into <b>linearized pBR322</b> for RIP expression and into <b>pSB1C3</b> for iGEM sample submission.<br>
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            <img src="https://static.igem.org/mediawiki/2018/4/46/T--Pasteur_Paris--PSB1C3_RIP.png">
 
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                <div class="legend"><b>Figure 12: </b> Agarose 1% gel after electrophoresis of digested pSB1C3 containing Seq8 (Bba_K2616001) with PstI and EcoRI. All colonies except 1, 3 and 7 contained the insert. </div>
 
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            <img src="https://static.igem.org/mediawiki/2018/9/95/T--Pasteur_Paris--_pBR322_RIP.png">
 
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              <div class="legend"><b>Figure 13: </b> Agarose 1% gel after electrophoresis of digested pBR322 containing Seq8 (Bba_K2616001) with NdeI (lane 1 to 7) All colonies except colonies 2 and 7 contained the insert.  </div>
 
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<p>We repeated the procedure (transformation in <i>E. coli</i> Stellar competent cells, bacteria culture, plasmid DNA extraction, digestion) and we proved that our vectors contained the insert by electrophoresis (Figure 12,13).<br>
 
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                <p>Alignment of <b>Sequencing</b> Results then confirmed that pSB1C3 contained Seq8, <a href="http://parts.igem.org/Part:BBa_K2616001"> Bba_K2616001 </a>. </p>
 
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                <img src="https://static.igem.org/mediawiki/2018/9/9f/T--Pasteur_Paris--SequencingRIP.PNG">
 
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                <div class="legend"><b>Figure 14: </b> Alignment of sequencing results for BBa_K2616001. Sequencing perform in pSB1C3 plasmid and one primer was designed (FOR1) to cover the whole sequence. Image from Geneious. Pairwise % Identity: 100%.  </div>
 
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                <div class="block full">
 
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                <p>Once checked, we cloned our construct into the <i>Escherichia coli</i> <b>BL21(DE3) pLysS</b> strain, a specific dedicated strain to produce high amounts of desired proteins under a T7 promoter. Bacteria were grown in 25 mL culture, and <b>protein expression</b> was induced with different IPTG concentration when bacteria have entered in a phase of exponential growth (approximately at 0.8 OD 600 nm) at 37°C. Pellet was sonicated and supernatant was kept<br>
 
−
                After two hours induction, we centrifuged and collect supernatant and pellet separately.<br><br></p></div>
 
−
 
−
 
−
        <div class="block full">
 
−
                <h4 style="text-align: left;">Fluorescence reading experiments</h4><br><br>
 
−
                <p>Since RIP is only a seven-aminoacid peptide, we were not able to check its production by classic SDS-PAGE. Thus, we tried to check its expression by <b>observing its effect</b> on <i>Staphylococcus aureus</i> growth and adhesion. We grew a <i>S. aureus</i> strain expressing GFP (Green Fluorescent Protein), gently provided by Dr. Jean-Marc Ghigo on 96-well microtiter plates with different fractions of supernatant or pellet of our BL21(DE3) pLysS bacterial cultures containing BBa_K26160001.<br><br></p>
 
−
<p>After 48h or more incubation, we washed the plates in order to discard planktonic bacteria, and read fluorescence (excitation at 485 nm and measuring emission at 510 nm).<br><br></p>
 
−
          </div>
 
−
 
−
            <div class="block half">
 
−
                <img src="https://static.igem.org/mediawiki/2018/d/dd/T--Pasteur_Paris--FluorescenceResults1.png">
 
−
                <div class="legend"><b>Figure 15: </b>Measurement of GFP fluorescence from <i>S. aureus</i> biofilms cultivated with different IPTG induction concentrations of RIP peptide. Every measure was done eight times and the bars show the average fluorescence. CM= Culture Medium from the induced <i>E. coli</i> culture.. SL = Lysis Supernatant from the induced <i>E. coli</i> culture.</div>
 
−
            </div>
 
−
 
−
        <div class="block half">
 
−
                <p>Some of the results we got were extremely encouraging. For example, figure 5 shows an average 3-fold reduction of fluorescence from <i>S. aureus</i> biofilms when they were cultivated in presence of the bacterial lysate of an induced culture of BL-21 <i>E. coli</i> transformed with  BBa_K2616001. </p>
 
−
                <p>However, we performed experiments several times, and the results were not always as concluding. This variability is very likely due to a bias due regarding different approaches used for supernatant removal and washes. When using the flicking approach, we damaged our biofilm. Then, we removed planktonic cells by micropipette. </p>
 
−
            </div>
 
−
 
−
        <div class="block full">
 
−
                <h4 style="text-align: left;">Crystal violet staining</h4><br><br>
 
−
                <p>Since fluorescence measurements were not satisfying enough, we tried to improve our methods to quantify biofilm formation. Thus, we began to color biofilms by Crystal violet 0.1% staining and measuring absorbance at 570 nm. Again, the results were very heterogeneous between our different experiments, and between the different protocols. For instance, we tried to compare our protocol to WPI Worcester team's staining protocol, and the results, given in Figue 6 and 7 significantly differ.</p>
 
−
          </div>
 
−
 
−
            <div class="block half">
 
−
                <img src="https://static.igem.org/mediawiki/2018/f/fa/T--Pasteur_Paris--CVPasteur.png">
 
−
            </div>
 
−
 
−
            <div class="block half">
 
−
                <img src="https://static.igem.org/mediawiki/2018/9/9f/T--Pasteur_Paris--CVWPI.png">
 
−
            </div>
 
−
 
−
<div class="block full">
 
−
<div class="legend"><b>Figure 16: </b>Measurement of absorbance at 570 nm <i>S. aureus</i> biofilms cultivated with different IPTG induction concentrations of RIP peptide and stained with Crystal violet. Every measure was done eight times and the bars show the average fluorescence. CM= Culture Medium from the induced <i>E. coli</i> culture.. SL = Lysis Supernatant from the induced <i>E. coli</i> culture.</div>
 
−
            </div>
 
−
 
−
 
−
        <div class="block two-third">
 
−
                <h4 style="text-align: left;">Biofilm PFA fixation before staining</h4><br><br>
 
−
<p>We wanted to avoid biofilm damage or loss during theses steps. In order to do that, we used Bouin solution to fix the formed biofilm after 24 and 48 hours of culture. Then biofilms were either stained with Crystal Violet 0.1% and resuspended in acetic acid 30% or resuspended in PBS 1X.  Surprisingly, with this method biofilm formation was higher when cultivated with cell extracts containing RIP. A that time, we are not able to explain why.</p>
 
−
</div>
 
−
 
−
            <div class="block one-third">
 
−
                <img src="https://static.igem.org/mediawiki/2018/f/f1/T--Pasteur_Paris--96-culture-wells-2.jpg">
 
−
                <div class="legend"><b>Figure 17: </b>Biofilm culture fixed with Bouin's solution in 96-well micrometer plate</div>
 
−
            </div>
 
−
 
−
 
−
<div class="block full">
 
−
<p>With more time, we would certainly have been able to optimize our protocols to best fit with the strain we use, but for the time being, we are not able to give a final conclusion on whether or not our RIP peptide inhibits <i>S. aureus</i> biofilm formation.
 
−
<br><br></p></div>
 
−
 
−
            <div class="block full">
 
−
            <h2><i>S. aureus</i></b> Detection and RIP secretion <a href="http://parts.igem.org/Part:BBa_K2616003"> BBa_K2616003</a></h2><br><br>
 
−
<p>The sequence we designed contains the <i> agr </i> detection system from <i>S. aureus</i> and secretion of  RIP (RNAIII Inhibiting Peptide) sequences fused to two different export signal peptides for <i>E. coli</i> Type II Secretion System: DsbA and MalE, placed in N-terminal.</p>
 
−
</div>
 
−
 
−
            <div class="block two-third center">
 
−
                <img src="https://static.igem.org/mediawiki/2018/9/92/T--Pasteur_Paris--BBa_K2616003.png">
 
−
                <div class="legend"><b>Figure 18: </b> <I> S. aureus </I> sensor device and RIP production cassette</div>
 
−
            </div>
 
−
 
−
<div class="block full">
 
−
<p>Once we received the sequence encoding for this production cassette, named Seq5 (1422 bp), Seq6 (960 bp) and Seq7 (762 bp) in commercial plasmid pEX-A258 by gene synthesis.  Plasmids was amplified in competent <i>E. coli</i> DH5alpha. <br><br>
 
−
After bacterial culture and plasmid DNA extraction, we digested the commercial vector with XbaI and BamHI for Seq5, MscI and SphI for Seq6, HindII and SpeI for Seq7 restriction enzymes. We extracted the insert from the gel and ligated by specific overlaps into linearized pBR322 for expression and into pSB1C3 for iGEM sample submission.</p>
 
−
 
−
<p>We had trouble to proceed the ligation of the three inserts to linearized pBR322 and pSB1C3. We discussed with Takara Bio about our ligation issues, the GC percentage on our overlaps was to high to allow a good ligation.  Due to the lack of time we were not able to re design the overlaps for this construction.  </p>
 
−
            </div>
 
−
 
−
 
 
             <div class="block separator-mark"></div>
 
             <div class="block separator-mark"></div>
  
 +
            <div class="block title" id="Design">
 +
                <h1>DESIGN</h1>
 +
        </div>
 +
          <div class="block two-third center">
 +
<p>We succeeded in conceptualizing an <b>ergonomic and functional</b> amputated <b>stump-to-prosthesis interface device</b> and charging station, at the scale of a prototype. We thought about its future design at an industrial scale.
 +
We also succeeded in <b>modeling in 3D</b> the entirety of the components of our device and of its charging station. We integrated a pragmatic and rapid <b>quarter turn system</b> for the osseointegrated stem so that patients are capable of putting on and taking off the device with ease.</p>
 +
<br>
 +
<p>We printed scale models using <b>stereolithographic techniques</b>. The thickness of the device's shell was first too substantial, which caused polymerization problems and tension between the two shells. We took into account those results, reduced the thickness and 3D-printed again the models, gaining in precision and lightness (-25% of the total weight). </p>
 +
<br>
 +
<p>The electronic schematics for the induction charging system, as well as the LED loop, allowing the <b>visualization</b> of charging and synchronizing of the device was also added to the device. The electronic parts are perfectly fitted into the 3D-printed device, and the charging system is totally operational.</p>
 +
<br>
 +
<p>We conceived a usage scenario, as well as a smartphone application.</p>
 +
<br>
 +
<p>To conclude, we tried to think of all the aspects of our device, from security, to manufacture, and ergonomy. We <b>demonstrated</b> a complete approach of product design incorporating a synthetic biology solution to nerve growth and biofilm infections in prostheses.</p>
  
−
            <div class="block title" id="Kill">
 
−
                <h1>KILL SWITCH</h1>
 
−
                <i style="text-align: left;"><p>Achievements:<br>
 
−
                    <ul>
 
−
                        <li>Successfully cloned a part coding for toxin/antitoxin (CcdB/CcdA) system in  iGEM plasmid backbone, creating a <b>new basic part</b> <a href="http://parts.igem.org/Part:BBa_K2616002"> Bba_K2616002 </a></li>
 
−
                        <li>Successfully sequenced  <a href="http://parts.igem.org/Part:BBa_K2616002"> BBa_K2616002</a> in pSB1C3 and sent to iGEM registry</li>
 
−
                        <li>Successfully observe survival of our engineered bacteria at 25°C and 37°C and <b>absence of growth</b> at 18°C and 20°C, showing the <b>efficiency of the kill switch</b></li>
 
−
                    </ul><br></p>
 
−
                    <p>Next steps:<br>
 
−
                    <ul>
 
−
                        <li>Find a system that kills bacteria when released in the environment rather than just stopping their growth</li>
 
−
                    </ul>
 
−
                </p></i>
 
−
            </div>
 
−
                <div class="block half">
 
−
                        <p>The sequence designed codes for two different proteins: <b>CcdB toxin</b> and <b>CcdA antitoxin</b>. The antitoxin production is under an constitutive promoter (PLac) and the toxin production under a thermosensitive one (PcspA).(Figure 19)</p>
 
−
            </div>
 
−
            <div class="block half">
 
−
          <img src="https://static.igem.org/mediawiki/2018/3/33/T--Pasteur_Paris--BBa_K2616002.png">
 
−
          <div class="legend"><b>Figure 19: </b>Cryo-death Kill-switch construction </div>
 
−
              </div>
 
  
 +
    </div>
  
 +
  <div class="block separator-mark"></div>
  
−
<div class="block half">
+
             <div class="block title" id="Entrepreneurship">
−
                <p>Once we received the sequence encoding for this production cassette, named Seq9 (981 bp) in commercial plasmid pEX-A258 by gene synthesis. Plasmids was amplified in competent <i>E. coli</i> DH5alpha.  After bacterial culture and plasmid DNA extraction, we digested the commercial vector with EcoRI and PstI restriction enzymes, extracted the insert from the gel, and ligated by specific overlaps into linearized pSB1C3 for iGEM submission and expression in BL21(DE3) pLysS.</p>
+
                 <h1>ENTREPRENEURSHIP</h1>
−
                <p>We repeated the procedure (transformation in <i>E. coli</i> Stellar competent cells, bacteria culture, plasmid DNA extraction, digestion) and we proved that our vector pSB1C3 contained Seq9 (Figure 20) after digestion and DNA electrophoresis. Plasmid DNA of pSB1C3 construction was purified and sent for sequencing.</p>
+
        </div>
−
            </div>
+
          <div class="block two-third center">
−
             <div class="block half">
+
<p>We successfully realized a <b>study target market analysis</b>, allowing us to visualize the population NeuronArch was aiming at and its evolution over the coming years. </p>
−
                <img src="https://static.igem.org/mediawiki/2018/4/4a/T--Pasteur_Paris--ClonageKS-pSB1C3.png">
+
<br>
−
                 <div class="legend"><b>Figure 20: </b> Agar gel after electrophoresis of digested pSB1C3 containing Seq9 (Bba_K2616002) in columns 6 to 11. Colonies 2 and 6 have the correct plasmid. </div>
+
−
            </div>
+
−
<div class="block full">
+
−
                <p>Alignment of <b>Sequencing</b> Results then confirmed that pSB1C3 contained Seq9, <a href="http://parts.igem.org/Part:BBa_K2616002"> Bba_K2616002 </a>. </p>
+
−
            </div>
+
−
            <div class="block two-third center">
+
−
                <img src="https://static.igem.org/mediawiki/2018/d/d1/T--Pasteur_Paris--Sequencing-KS.PNG">
+
−
                <div class="legend"><b>Figure 21: </b> Alignment of sequencing results for BBa_K2616002. Sequencing perform in pSB1C3 and two primers were designed (FOR1 and FOR2) to cover the whole sequence. Image from Geneious. Pairwise Identity: 96.9%.  </div>
+
−
            </div>
+
  
−
<div class="block full">
+
<p>We studied and analyzed our possible <b>competitors</b>, such as Ossur, Integrum, Ottobock, etc. and analyzed our <b>advantages</b> compared to them. We designed a marketing plan, showed our sales plan and communication strategy. We also talked about our current (and possible future) <b>sponsors and collaborators</b>, as well as the crowdfunding. </p>
−
                <p>The construction was successfully assembled. On Figure 21, mismatches are visible which correspond to the reduced precision of sequencing after 600 bp. To avoid this lack of precision, we used two different primers, allowing us to cover the whole sequence without mistakes. As visible, the mismatches are only present at the extremities of each primer sequencing. </p>
+
<br>
−
            </div>
+
<p>We also showed how we managed our <b>budget</b> during this iGEM competition to bring all 19 team members and 4 coaches to Boston in addition to our lab expenses, and how we plan to budget in the future. </p>
 +
<br>
  
−
            <div class="block full">
+
    </div>
−
                <p>To test the efficiency of our kill-switch, we decided to cultivate BL21(DE)3 E. coli transformed with it at several temperatures (15°C, 20°C, 25°C and 37°C). The growth was followed by measuring the optical density at 600nm every 30 minutes for 6 hours, followed by two additional points at 18 hours and at 72 hours. Each experiment was done in a triplicate and the standard deviations were calculated for every point.  We show that the bacteria transformed with the kill-switch showed <b>no measurable growth</b> at 15°C and at 20°C during the 72 hours of the experiment, whereas the control population grew normally.</p>
+
−
                <p>At 25°C, the kill-switch population grew more slowly than the control for the first 18 hours, but the growth eventually started to reach normal values at 72 hours. </p>
+
−
                <p>Finally, at 37°C there was no difference in the growth of the kill-switch population compared to the control bacteria. </p>
+
−
            </div>
+
−
            <div class="block two-third center">
+
−
                <img src="https://static.igem.org/mediawiki/2018/1/1b/T--Pasteur_Paris--kill-switch-graph-no-title.png">
+
−
                <div class="legend"><b>Figure 20: </b>Effect of different temperatures on the growth of Cryodeath kill-switch transformed BL21 <i>E. coli</i></div>
+
−
            </div>
+
−
            <div class="block full">
+
−
                <p>Thus, we successfully guarantee that our engineered bacteria will not be able to grow if they happened to be released in the environment.</p>
+
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Latest revision as of 14:24, 10 November 2018

""

NERVE GROWTH FACTOR AND NEURONAL CULTURE

We successfully designed and cloned a biobrick coding for the secretion of rat proNGF in pET43.1a and iGEM plasmid backbone pSB1C3, creating the new part Bba_K2616000 . We confirmed this genetic construct by sequencing.


We confirmed the production of proNGF in three different ways:

  • SDS PAGE followed by western blot.
  • Mass spectrometry.
  • Activity on the growth of rat E18 cortical cells.

We designed and manufactured microfluidic chips in order to test our final proof of concept.

We grew embryonic E18 rat neurons in our self-made microfluidic chips and successfully observed axon growth in the presence of commercial NGF and our recombinant proNGF.

To conclude, we showed that the activity of our recombinant proNGF was comparable to the one of commercial NGF used at concentrations of 500 to 900 ng/mL, demonstrating that our theoretical idea works in vitro.

KILL SWITCH

We successfully designed and cloned a biobrick coding for a temperature sensitive kill-switch, creating the new part Bba_K2616002 . We confirmed this genetic construct by sequencing.


We observed the survival and normal growth of our engineered chassis E. coli BL21(DE3)pLysS at 25°C and 37°C and the absence of growth at 18°C and 20°C. Comparatively, our negative control, the same chassis, transformed with an empty vector, grew normally at all temperatures tested.


To conclude, we have demonstrated that our kill-switch is a very efficient way to prevent the contamination of the environment by our genetically modified bacteria in case of accidental release.

MEMBRANE BIOCOMPATIBILITY AND CONDUCTIVITY

In search of a biocompatible conductive polymer to confine bacteria, we successfully polymerized PEDOT:Ts and PEDOT:Cl on alumina oxyde membrane filters. We also partially polymerized PEDOT:PSS.


We demonstrated that a polymer-coating enhances the electrical properties of the membranes as PEDOT:Ts-coated and PEDOT:Cl-coated membranes are more conductive than uncoated membranes. Moreover, experiments showed a better biocompatibility for the polymer-coated membranes compared to the gold-coated ones.


To conclude, we successfully demonstrated that our membrane could confine bacteria. This feature is one of the essential safety components that we have engineered in our NeuronArch project, ensuring the patient's safety.

DESIGN

We succeeded in conceptualizing an ergonomic and functional amputated stump-to-prosthesis interface device and charging station, at the scale of a prototype. We thought about its future design at an industrial scale. We also succeeded in modeling in 3D the entirety of the components of our device and of its charging station. We integrated a pragmatic and rapid quarter turn system for the osseointegrated stem so that patients are capable of putting on and taking off the device with ease.


We printed scale models using stereolithographic techniques. The thickness of the device's shell was first too substantial, which caused polymerization problems and tension between the two shells. We took into account those results, reduced the thickness and 3D-printed again the models, gaining in precision and lightness (-25% of the total weight).


The electronic schematics for the induction charging system, as well as the LED loop, allowing the visualization of charging and synchronizing of the device was also added to the device. The electronic parts are perfectly fitted into the 3D-printed device, and the charging system is totally operational.


We conceived a usage scenario, as well as a smartphone application.


To conclude, we tried to think of all the aspects of our device, from security, to manufacture, and ergonomy. We demonstrated a complete approach of product design incorporating a synthetic biology solution to nerve growth and biofilm infections in prostheses.

ENTREPRENEURSHIP

We successfully realized a study target market analysis, allowing us to visualize the population NeuronArch was aiming at and its evolution over the coming years.


We studied and analyzed our possible competitors, such as Ossur, Integrum, Ottobock, etc. and analyzed our advantages compared to them. We designed a marketing plan, showed our sales plan and communication strategy. We also talked about our current (and possible future) sponsors and collaborators, as well as the crowdfunding.


We also showed how we managed our budget during this iGEM competition to bring all 19 team members and 4 coaches to Boston in addition to our lab expenses, and how we plan to budget in the future.