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<span class="closeCross"><img src="https://static.igem.org/mediawiki/2018/6/67/T--Pasteur_Paris--CloseCross.svg"></span> | <span class="closeCross"><img src="https://static.igem.org/mediawiki/2018/6/67/T--Pasteur_Paris--CloseCross.svg"></span> | ||
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− | + | <div class="block title" id="Fight"> | |
− | + | <h1>FIGHT INFECTIONS</h1><br> | |
− | + | </div> | |
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+ | <div class="block full"> | ||
+ | <h2>RIP Secretion <a href="http://parts.igem.org/Part:BBa_K2616001"> BBa_K2616001</a></h2><br><br> | ||
+ | |||
+ | <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> | ||
+ | <div class="block two-third center"> | ||
+ | <img src="https://static.igem.org/mediawiki/2018/f/fd/T--Pasteur_Paris--BBa_K2616001.png"> | ||
+ | <div class="legend"><b>Figure 11: </b>proNGF and TEV production cassette </div> | ||
+ | </div> | ||
+ | <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> | ||
+ | |||
+ | </div> | ||
+ | <div class="block half"> | ||
+ | <img src="https://static.igem.org/mediawiki/2018/4/46/T--Pasteur_Paris--PSB1C3_RIP.png"> | ||
+ | <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> | ||
+ | </div> | ||
+ | <div class="block half"> | ||
+ | <img src="https://static.igem.org/mediawiki/2018/9/95/T--Pasteur_Paris--_pBR322_RIP.png"> | ||
+ | <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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</div> | </div> | ||
− | <div class="block | + | <div class="block full"> |
− | <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 | + | <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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</div> | </div> | ||
+ | <div class="block full"> | ||
+ | <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> | ||
+ | </div> | ||
+ | <div class="block two-third center"> | ||
+ | <img src="https://static.igem.org/mediawiki/2018/9/9f/T--Pasteur_Paris--SequencingRIP.PNG"> | ||
+ | <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> | ||
+ | </div> | ||
+ | <div class="block full"> | ||
+ | <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> | ||
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− | + | <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> | |
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<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> | <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> | |
− | + | ||
− | <p> | + | <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/2018/7/77/T--Pasteur_Paris--ImproveParts.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> | ||
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<div class="block separator-mark"></div> | <div class="block separator-mark"></div> | ||
</div> | </div> | ||
<div class="block full" style="background-color: transparent;"> | <div class="block full" style="background-color: transparent;"> | ||
− | + | <i style="text-align: left;"><p>Achievements:<br> | |
− | + | <ul> | |
− | + | <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>. | |
− | + | <li>Successfully sequenced <a href="http://parts.igem.org/Part:BBa_K2616001"> Bba_K2616001 </a> in pSB1C3 and sent to iGEM registry. | |
− | + | <li>Successfully cultivated S. aureus biofilms in 96 well plates with different supernatants.</li> | |
− | + | </ul><br></p> | |
− | + | <p>Next steps:<br> | |
− | + | <ul> | |
− | + | <li>Clone the sensor device with inducible RIP production upon S. aureus detection.</li> | |
− | + | <li>Improve the characterization of RIP effect on biofilm formation.</li> | |
− | + | </ul> | |
+ | </p></i> | ||
</div> | </div> |
Revision as of 22:32, 15 October 2018
FIGHT INFECTIONS : Click to see more
Achievements:
- Successfully cloned a part coding for RIP secretion in pBR322 and in pSB1C3, creating a new part Bba_K2616001 .
- Successfully sequenced Bba_K2616001 in pSB1C3 and sent to iGEM registry.
- Successfully cultivated S. aureus biofilms in 96 well plates with different supernatants.
Next steps:
- Clone the sensor device with inducible RIP production upon S. aureus detection.
- Improve the characterization of RIP effect on biofilm formation.
RECONNECT NERVES: Click to see more
Achievements:
- Successfully cloned a part coding for secretion of NGF in pET43.1a and iGEM plasmid backbone pSB1C3, creating a new composite part BBa_K2616000
- Successfully sequenced BBa_K2616000 in pSB1C3 and sent to iGEM registry
- Successfully co-transform E. coli with plasmid secreting NGF and plasmid expressing the secretion system, creating bacteria capable of secreting NGF in the medium
- Successfully characterized production of NGF thanks to mass spectrometry
- Successfully observe axon growth in microfluidic chip in presence of commercial NGF
Next steps:
- Purify secreted NGF, and characterize its effects on neuron growth thanks to our microfluidic device
- Global proof of concept in a microfluidic device containing neurons in one of the chamber, and our engineered bacteria in the other
KILL SWITCH: Click to see more
Achievements:
- Successfully cloned a part coding for toxin/antitoxin (CcdB/CcdA) system in iGEM plasmid backbone, creating a new composite part
- Successfully observe survival of our engineered bacteria at 25°C and 37°C and absence of growth at 18°C and 20°C, showing the efficiency of the kill switch
Next steps:
- Find a system that kills bacteria when released in the environment rather than just stopping their growth