Difference between revisions of "Team:Pasteur Paris/TrainingThomas2"

Revision as of 17:38, 16 October 2018

Reconnect Nerves

Fight Infections

Kill Switch

Membrane

RECONNECT NERVES

DNA assembly

The sequence we designed codes for two different proteins: proNGF (Nerve Growth Factor) and TEV protease (from Tobacco Etch Virus). These two proteins are fused in C-terminal with a signal peptide for E. coli Type I Secretion System which consists in the last 60 amino-acids of HaemolysinA (HlyA). 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).

Figure 1: proNGF and TEV production cassette

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 E. coli DH5-α. After bacteria culture and plasmid DNA extraction, we digested commercial vectors with restriction enzymes (NheI and BamHI for Seq1, MscI and HindIII for Seq2). We extracted the inserts from the gel and performed a ligation by using specific overlaps into linearized pET43.1a for proNGF expression and into pSB1C3 for iGEM sample submission.
We repeated the procedure (transformation in E. coli 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).

Figure 2: 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.

Figure 3: 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.

Alignment of Sequencing Results then confirmed that pSB1C3 contained Seq1 and Seq2, BBa_K2616000 .

Figure 4: 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.

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.

proNGF characterization and purification

Our chassis is Escherichia coli BL21(DE3) pLysS, a specific strain dedicated to producing high amounts of desired proteins under a T7 promoter. Thus, we co-transformed our bacteria with BBa_K2616000 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.

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.

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.

Figure 5: 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.

We analyzed bacterial lysate and purification fractions using SDS-PAGE electrophoresis and Mass spectrometry.

Figure 6: SDS-PAGE gel Bis-Tris 4-12% of bacterial lysate and proNGF purification fraction by SDS-PAGE.

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.

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.

Figure 7: Distribution of proNGF and TEV protease by gel fractions after mass spectrometry analysis.

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

Figure 8: 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.

Figure 9: Mass spectrometry spectrum. A) Peptide identified corresponding to proNGF. B) Peptide identified corresponding to the fusion of proNGF and HlyA export signal.

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.

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.

Figure 10: Western Blot analysis of batch purification proNGF under native and partial denaturing conditions.

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

FIGHT INFECTIONS

RIP Secretion BBa_K2616001

The sequence we designed contains two RIP (RNAIII Inhibiting Peptide) sequences fused to two different export signal peptides for E. coli Type II Secretion System: DsbA and MalE, 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).)

Figure 11: proNGF and TEV production cassette

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 E. coli DH5alpha. After bacteria culture and plasmid DNA extraction, we digested commercial vector with EcoRI and PstI restriction enzymes. We extracted the inserts from the gel and performed a ligation by using specific overlaps into linearized pBR322 for RIP expression and into pSB1C3 for iGEM sample submission.

Figure 12: 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.

Figure 13: 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.

We repeated the procedure (transformation in E. coli Stellar competent cells, bacteria culture, plasmid DNA extraction, digestion) and we proved that our vectors contained the insert by electrophoresis (Figure 12,13).

Alignment of Sequencing Results then confirmed that pSB1C3 contained Seq8, Bba_K2616001 .

Figure 14: 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%.

Once checked, we cloned our construct into the Escherichia coli BL21(DE3) pLysS strain, a specific dedicated strain to produce high amounts of desired proteins under a T7 promoter. Bacteria were grown in 25 mL culture, and protein expression 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
After two hours induction, we centrifuged and collect supernatant and pellet separately.

Fluorescence reading experiments

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 observing its effect on Staphylococcus aureus growth and adhesion. We grew a S. aureus 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.

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).

Figure 15: Measurement of GFP fluorescence from S. aureus 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 E. coli culture.. SL = Lysis Supernatant from the induced E. coli culture.

Some of the results we got were extremely encouraging. For example, figure 5 shows an average 3-fold reduction of fluorescence from S. aureus biofilms when they were cultivated in presence of the bacterial lysate of an induced culture of BL-21 E. coli transformed with BBa_K2616001.

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.

Crystal violet staining

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.

Figure 16: Measurement of absorbance at 570 nm S. aureus 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 E. coli culture.. SL = Lysis Supernatant from the induced E. coli culture.

Biofilm PFA fixation before staining

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.

Figure 17: Biofilm culture fixed with Bouin's solution in 96-well micrometer plate

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 S. aureus biofilm formation.

S. aureus Detection and RIP secretion BBa_K2616003

The sequence we designed contains the agr detection system from S. aureus and secretion of RIP (RNAIII Inhibiting Peptide) sequences fused to two different export signal peptides for E. coli Type II Secretion System: DsbA and MalE, placed in N-terminal.

Figure 18: S. aureus sensor device and RIP production cassette

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 E. coli DH5alpha.

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.

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.

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.

KILL SWITCH

Once we received the sequences encoding for this production cassette (named construction Seq9) in commercial plasmids, in order to have more DNA, we transformed competent bacteria E. coli DH5alpha resulting in clones. After bacteria culture and plasmid DNA extraction, we digested commercial vectors with restriction enzymes, extracted the inserts from the gel, and ligated it into linearized pSB1C3 for iGEM submission and expression in BL21(DE)3.

We repeated the procedure (transformation in E. coli Stellar competent cells, bacteria culture, plasmid DNA extraction, digestion) and we proved that our vector contained the insert by electrophoresis.

Figure 7: Agar gel after electrophoresis of digested pSB1C3 containing Seq9 (Bba_K2616002) in columns 6 to 11. Colonies 2 and 6 have the correct plasmid.

Alignment of Sequencing Results then confirmed that pSB1C3 contained Seq9, Bba_K2616002 .

Figure 21: 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%.

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.

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 no measurable growth at 15°C and at 20°C during the 72 hours of the experiment, whereas the control population grew normally.

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.

Finally, at 37°C there was no difference in the growth of the kill-switch population compared to the control bacteria.

Figure 22: Effect of different temperatures on the growth of Cryodeath kill-switch transformed BL21 E. coli

Thus, we successfully guarantee that our engineered bacteria will not be able to grow if they happened to be released in the environment.

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

Membrane

The membrane filter is a key element of our prosthesis system, allowing the confinement of the genetically modified bacteria and the conduction of neuron impulses. We tested two types of membranes: Sterlitech Polycarbonate Gold-Coated Membrane Filters (pores diameter of 0.4 micrometer) and Sterlitech Alumina Oxide Membrane Filters (pores diameter of 0.2 micrometer).
Sterlitech Alumina Oxide Membrane Filters were coated with different types of biocompatible conductive polymers: PEDOT:PSS (poly(3,4-ethylenedioxythiophene) polystyrene sulfonate), PEDOT:Cl and PEDOT:Ts.
To characterize the potential of the different types of membranes to be integrated in our prosthesis system, we evaluated the coating of the alumina oxide membranes, their biocompatibility and their electrical conductivity.

Figure 23: PEDOT:PSS

Biocompatibility

Conductivity

Figure 24: Hand-made PDMS well chip

The conductivity of the membranes was measured on a self-made device. It consists of a culture well made of PDMS (polydimethylsiloxane), with a membrane filter at its bottom and a platinum wire linking the conductive membrane filter with the exterior.

Platinum wire

The voltage difference between the two extremities of the wire was measured.

Figure 25: Voltage difference between the two extremities of the platinum wire.

The voltage difference between different platinum wires is pretty much the same. As we want to compare the differences between multiple membranes, we don't need to take into account the variability from one chip to another of the platinum wire's resistance. That means, it is meaningful to measure the voltage difference between a point on the membrane and the extremity of the wire outside the well, and use this data to compare the membranes.

Membranes

The voltage difference between a point on the membrane (located near the border of the membrane filter) and the extremity of the platinum wire outside the well was measured.

Figure 26: Voltage difference between the extremity of the platinum wire outside the well and a point on the membrane.

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-	/* BEGIN : Fractions Styles */
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+  <div id="bannerchanged">
+                 <img class="banner-img" src="https://static.igem.org/mediawiki/2018/6/64/T--Pasteur_Paris--Banner_Results.jpg">
+        </div>
+    <h1></h1>
+  </div>
-	<div id="banner">
+  <div id="GeneralContent">
-		<h1>MODELING</h1>
+    <div id="index" class="block">
-	</div>
+      <div id="indexContent">
+        <p><a href="#Reconnect" class="link">Reconnect Nerves</a></p>
+        <p><a href="#Fight" class="link">Fight Infections</a></p>
+        <p><a href="#Kill" class="link">Kill Switch</a></p>
+        <p><a href="#Membrane" class="link">Membrane</a></p>
+      </div>
+      <div id="indexRight">
+         <div id="littleButton"></div>
+      </div>
+    </div>
+    <div id="MainContent">
+ <!-- First Onglet Reconnect nerves-->
+                <div class="block full bothContent">
+                    <div class="block dropDown" id="Reconnect">
+                        <h4>RECONNECT NERVES: <span>Click to see more</span></h4>
+                    </div>
-	<div id="GeneralContent">
+                    <div class="block hiddenContent">
-		<div id="index" class="block">
+                        <span class="closeCross"><img src="https://static.igem.org/mediawiki/2018/6/67/T--Pasteur_Paris--CloseCross.svg"></span>
-			<div id="indexContent">
+           <div class="block title">
-				<p><a href="#Introduction" class="link">Introduction</a></p>
+                <h1>RECONNECT NERVES</h1>
-				<p><a href="#Production" class="link">NGF Production</a></p>
+            </div>
-				<p><a href="#Diffusion" class="link">NGF Diffusion</a></p>
-				<p><a href="#Growth" class="link">Neurons Growth</a></p>
-				<p><a href="#References" class="link">References</a></p>
-			</div>
-			<div id="indexRight">
-				 <div id="littleButton"></div>
-			</div>
-		</div>
-		<div id="MainContent">
-			<!-- Introduction -->
-				<div class="block title" id="Introduction">
-					<h1>General introduction</h1>
-				</div>
-				<div class="block two-third">
-					<p>The aim of our mathematical model is to simulate the growth of neurons towards our biofilm in response of the presence of Nerve Growth Factor (NGF). Nerve growth factor is one of a group of small proteins called neurotrophins that are re-sponsible for the development of new neurons, and for the health and maintenance of mature ones. We created a determin-istic model to help the wetlab establish the optimal concen-tration gradients of NGF needed for the regrowth of the nerves. NGF concentration and concentration gradient are key parameters affecting the growth rate and direction of neu-rites. Neurites growth have shown to be NGF dose-dependent: if NGF concentration si too low or too high, the growth rate is attenuated. In order to visualize the results of the model on a microfluidic chip we used MATLAB, App Designer, Python, Gmsh and FreeFem. This is an important part of our project since it creates the link between the wetlab and drylab. </p>
-				</div>
-				<div class="block one-third">
-					<img src="https://static.igem.org/mediawiki/2018/2/23/T--Pasteur_Paris--neurone%2BNGF%2Bchip.png" style="max-width: 450px">
-				</div>
-				<div class="block full">
-					<p style="text-align: center;">We divided our model in three parts:
-					<ol style="text-align: left;">
-						<li>Production of NGF by the <i>E. coli</i> genetically modified</li>
-						<li>Simulation of the diffusion of NGF in a given environment</li>
-						<li>Neurons growth in the presence of NGF</li>
-					</ol>
-					</p>
-				</div>
-				<div class="block separator-mark"></div>
-				<div class="block title">
-					<h1>Context of our model</h1>
-				</div>
-				<div class="block half">
-					<p>Our project aims at creating a biofilm composed of genetically modified <i>E. coli</i> able to release a neurotrophic factor: NGF. It helps to accelerate the connection between the neurons and the implant of the prothesis; hence aiming at connecting directly the prothesis amputee’s neurons. This will enable the patient to have a more instinctive control of his prothesis device. The nerves will be guided towards a conductive membrane surrounding our genetically modified biofilm. This membrane will then pass the neural signal of the regenerated nerves towards the electronic chip of the implant through wires.  It will allow the patient to have a more instinctive and natural control than any other current prosthesis, and a reduced reeducation time.</p>
-				</div>
-				<div class="block half">
-					<img src="https://static.igem.org/mediawiki/2018/d/d4/T--Pasteur_Paris--membrane-plus-egal.png">
-				</div>
-				<div class="block one-third">
-					<img src="https://static.igem.org/mediawiki/2018/b/b7/T--Pasteur_Paris--micropuce.png" style="max-width: 300px">
-				</div>
-				<div class="block two-third">
-					<p>The aim of the wetllab is to test the biofilm on a microfluidic chip as a proof of concept. The chip is composed of two compartments: one made of the E. coli genetically modified to produce NGF and the other one of neurons. Micro canals link the two compartments in the middle of the chip, allowing the diffusion of NGF and the growth of the neurites. 	Our model will hence be established on a micro-fluidic chip shape in order to share our results with the wetlab and indicate them the optimal concentration of NGF needed according to their model.</p>
-				</div>
-				<div class="block two-third center">
-					<p><i>We introduce different parameters in order to create our model :</i></p>
-					<table class="tableData" style="margin: auto;">
-						<tr>
-							<td>g</td>
-							<td>Length of the neurite outgrowth</td>
-						</tr>
-						<tr>
-							<td>
-								<span>
-									<div class="frac"><span>dg</span><span class="symbol">/</span><span class="bottom">dt</span></div>
-								</span>
-							</td>
-							<td>Neurite outgrowth rate</td>
-						</tr>
-						<tr>
-							<td>u(x,t)</td>
-							<td>Concentration of NGF at the position x and time t</td>
-						</tr>
-						<tr>
-							<td>
-								<span>
-									<div class="frac"><span>du</span><span class="symbol">/</span><span class="bottom">dt</span></div>
-								</span>
-							</td>
-							<td>NGF concentration gradient at the position x and time t</td>
-						</tr>
-						<tr>
-							<td>C<SUB>diff</SUB></td>
-							<td>Diffusion coefficient of NGF</td>
-						</tr>
-						<tr>
-							<td>K</td>
-							<td>Gradient factor (growth rate of the neurite under the stimulation of the NGF concentration gradient)</td>
-						</tr>
-						<tr>
-							<td>G<SUB><FONT face="Raleway">&theta;</FONT></SUB></td>
-							<td>Baseline growth rate (neurite growth rate in absence of NGF concentration gradient)</td>
-						</tr>
-						<tr>
-							<td>L</td>
-							<td>Length of the conduit </td>
-						</tr>
-					</table>
-				</div>
-				<div class="block separator"></div>
-			<!-- First Onglet Production of NGF-->
+        <div class="block half">
-				<div class="block full bothContent">
+                <h4 style="text-align: left;">DNA assembly</h4><br><br>
-					<div class="block dropDown" id="Production">
+                <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>
-						<h4>NGF Production by genetically modified <i>E. coli</i></h4>
+            </div>
-					</div>
+            <div class="block half">
+                <img src="https://static.igem.org/mediawiki/2018/8/80/T--Pasteur_Paris--ProNGFandTEVproduction.png">
+                <div class="legend"><b>Figure 1: </b>proNGF and TEV production cassette </div>
+            </div>
+            <div class="block full">
+                <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>
+            </div>
+            <div class="block half">
+                <img src="https://static.igem.org/mediawiki/2018/7/7c/T--Pasteur_Paris--gelNGFpET.png">
+                <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>
+</div>
+             <div class="block half">
+                <img src="https://static.igem.org/mediawiki/2018/e/ef/T--Pasteur_Paris--gelNGFpSB1C3.png">
+                <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>
+            </div>
-					<div class="block hiddenContent">
-						<span class="closeCross"><img src="https://static.igem.org/mediawiki/2018/6/67/T--Pasteur_Paris--CloseCross.svg"></span>
-						<div class="block title">
-							<h1 style="padding-top: 50px;">NGF Production by genetically modified <i>E. coli</i></h1>
-							<p><i>As we want to obtain the best fitted NGF concentration, we first simulate the production and secretion of our recombinant NGF by transformed E. coli, in order to help the wetlab to optimize the induction and obtain the desired concentration, and to check whether we can theoretically obtain the optimal concentration for neurite growth.</i></p>
-						</div>
-						<div class="block full">
-							<h3>Model Description</h3>
-							<p>In this model, we include transcription, translation, translocation through E. coli  membrane, protein folding and mRNA and protein degradation in cytoplasm and medium. NGF synthesis is placed under Plac promoter, so we also modelled the IPTG induction. Finally, NGF is secreted to the medium through Type I secretion system in which the export signal peptide is not cleaved during translocation. Our Biobrick is design to synthetize and export TEV protease in order to cleave signal peptide and thus produce functional NGF.</p>
-							<p>The molecular mechanism included in our model appears schematically in:</p>
-						</div>
-						<div class="block two-third">
-						</div>
-						<div class="block full">
-							<p style="">Our model includes the following variables:</p>
-							<table class="tableData" style="margin: auto;">
-								<tr>
-									<td><b>Name</b></td>
-									<td><b>Meaning</b></td>
-								</tr>
-								<tr>
-									<td><b>I<sub>ex</sub></b></td>
-									<td>IPTG outside the cell</td>
-								</tr>
-								<tr>
-									<td><b>I<sub>in</sub></b></td>
-									<td>IPTG in the cytoplasm</td>
-								</tr>
-								<tr>
-									<td><b>P<sub>o</sub></b></td>
-									<td><i>Plac</i> promoter occupied by repressor, prevent transcription</td>
-								</tr>
-								<tr>
-									<td><b>P<sub>f</sub></b></td>
-									<td><i>Plac</i> promoter with free <i>lacO</i> site</td>
-								</tr>
-								<tr>
-									<td><b>m</b></td>
-									<td>mRNA for TEV and NGF</td>
-								</tr>
-								<tr>
-									<td><b>m-r</b></td>
-									<td>Ribosome-bound mRNA </td>
-								</tr>
-								<tr>
-									<td><b>NGF<sub>c</sub></b></td>
-									<td>NGF in cytoplasm</td>
-								</tr>
-								<tr>
-									<td><b>TEV<sub>c</sub></b></td>
-									<td>TEV protease in cytoplasm</td>
-								</tr>
-								<tr>
-									<td><b>(N-T)<sub>c</sub></b></td>
-									<td>NGF-TEV complex in cytoplasm</td>
-								</tr>
-								<tr>
-									<td><b>NGF<sub>cc</sub></b></td>
-									<td>Cleaved NGF in cytoplasm, cannot be exported</td>
-								</tr>
-								<tr>
-									<td><b>NGF<sub>t</sub></b></td>
-									<td>NGF bound to transporter channel</td>
-								</tr>
-								<tr>
-									<td><b>TEV<sub>t</sub></b></td>
-									<td>TEV bound to transporter channel</td>
-								</tr>
-								<tr>
-									<td><b>t</b></td>
-									<td>Transmembrane transporter</td>
-								</tr>
-								<tr>
-									<td><b>NGF<sub>um</sub></b></td>
-									<td>Unfolded NGF in medium with export peptide</td>
-								</tr>
-								<tr>
-									<td><b>NGF<sub>m</sub></b></td>
-									<td>Folded NGF in medium with export peptide</td>
-								</tr>
-								<tr>
-									<td><b>N-T<sub>m</sub></b></td>
-									<td>Complex between NGF with export peptide and functional TEV</td>
-								</tr>
-								<tr>
-									<td><b>TEV<sub>m</sub></b></td>
-									<td>TEV in medium with export peptide</td>
-								</tr>
-								<tr>
-									<td><b>NGF<sub>f</sub></b></td>
-									<td>Functional NGF in the medium</td>
-								</tr>
-							</table>
-						</div>
-						<div class="block separator"></div>
-						<div class="block title">
-							<h4 style="text-align: left;">1. NGF and TEV synthesis in the cytoplasm</h4>
-						</div>
-						<div class="block full">
-							<p>The synthesis of NGF and TEV is placed under the control of Plac promoter. The promoter can be in two different states: occupied (Po) by the repressor lacI, preventing RNA polymerase from binding and thus preventing transcription, or free (Pf) thanks to IPTG binding to the repressor. We assume that one IPTG molecule binds with one repressor molecule, freeing the promoter and restoring RNA polymerase binding capacity. The real mechanism of promoter Plac is more complex, as described in [1], but this simplification is sufficient for our model.</p>
-							<img src="">
-							<p>The transport of IPTG from outside the cell to cytoplasm is considered to be only due to free diffusion through the membrane by two first order reaction with the same kinetic constant.<p>
-							<img src="">
-							<p>IPTG is not considered to be degraded neither in the cytoplasm nor in the medium.</p>
-							<p>For the TEV and NGF transcription, we use a first-order reaction where the rate of mRNA production (m) depends on the concentration of the free promoter (Pf).</p>
-							<img src="">
-							<p>For the TEV and NGF translation, we first consider binding of ribosomes to ribosome binding site (the same association constant is used since the r.b.s. are the same), and then translation rate is proportional to the protein length. Since TEV and NGF have approximately the length, we consider only one translation rate β.</p>
-							<img src="">
-							<p>Even though it still has an export peptide, TEV is assumed to be functional in the cytoplasm (although less functional than if it had no export peptide). Since NGF has TEV cleaving site between the coding sequence and the export peptide, a fraction of NGF is cleaved inside the cytoplasm and thus cannot be secreted. We use a simple model to simulate TEV kinetics: TEV recognizes the signal sequence ENLYFQ, bind to its substrate and then cleave the export peptide. This process can thus be modeled by the following equations:</p>
-							<img src="">
-							<p>K1, k-1 and k2 are taken lower than constants found in literature, in order to model the fact that TEV still has its signal peptide and is consequently less functional than usually.</p>
-						</div>
-						<div class="block separator"></div>
-						<div class="block title">
-							<h4 style="text-align: left;">2. NGF and TEV secretion to the medium</h4>
-						</div>
-						<div class="block full">
-							<p>The transport of NGF and TEV with their export signal peptide from inside the cell to the medium is assumed to follow Michaelis-Menten enzymatic kinetics in which the transporter channel (composed of HlyB in the inner membrane, bound to HlyD and recruiting TolC in the outer membrane) plays the role of the enzyme and intracellular protein the role of the substrate.</p>
-							<p>Each protein (NGF and TEV) via its export signal peptide HlyA can bind to the HlyB-HlyD complex pore, forming a protein-transporter complex (NGFt or TEVt). Translocation correspond to the dissociation of this complex, resulting in restoring a free transporter and secreting NGF or TEV in the medium (NGFum and TEVm), which stand for the products.</p>
-							<img src="">
-						</div>
-						<div class="block separator"></div>
-						<div class="block title">
-							<h4 style="text-align: left;">3. Including growth rate</h4>
-						</div>
-						<div class="block full">
-							<p>This model stands for one bacterial cell, but for our model to best fit what our future system will look like, we need to integrate the growth rate of our bacteria within the microchannel chip well. Therefore, we measured DO of a culture of our bacteria, in stationary growth phase, in order to fit a growth equation.</p>
-							<p>For the growth rate of our transformed bacteria we use the equation fitted to the determined values of OD600. </p>
-							<img src="">
-						</div>
-						<div class="block separator"></div>
-						<div class="block title">
-							<h4 style="text-align: left;">4. NGF folding and export peptide cleavage by TEV</h4>
-						</div>
-						<div class="block full">
-							<p>Once in the medium, both NGF and TEV are still bound to the export signal peptide HlyA. We assume there is a very small amount of functional TEV, that is sufficient to cleave TEV signal peptide, producing more functional TEV.</p>
-							<p>As for the transporter, we use a simple model in which TEV recognizes the signal sequence ENLYFQ, bind to its substrate (which can be either NGF with its export peptide or TEV with its export peptide) and then cleave the export peptide. This process can thus be modeled by the following equations:</p>
-							<img src="">
-						</div>
-						<div class="block separator"></div>
-						<div class="block title">
-							<h4 style="text-align: left;">5. mRNA and protein degradation</h4>
-						</div>
-						<div class="block full">
-							<p>Finally, in cytoplasm and in the medium, mRNA and protein are degraded and all degradations are assumed to follow first-order kinetic reactions.</p>
-							<img src="">
-						</div>
-						<div class="block full">
-							<h3>MODEL PARAMETRISATION</h3>
-							<p>From these equations, we obtained a system of differential equations mostly based on mass action kinetics (GET IT HERE). We numerically solveD the ordinary differential equations system using Euler method implemented in Python. The constants we used were mainly determined from literature AND are given in table …</p>
-							<table class="tableData" style="margin: auto;">
-								<tr>
-									<td><b>NAME</b></td>
-									<td><b>DESCRIPTION</b></td>
-									<td><b>VALUE</b></td>
-									<td><b>UNIT</b></td>
-									<td><b>SOURCE</b></td>
-								</tr>
-								<tr>
-									<td>k<sub>t</sub></td>
-									<td>IPTG diffusion rate across the membrane</td>
-									<td>0.92</td>
-									<td>min<sup>-1</sup></td>
-									<td></td>
-								</tr>
-								<tr>
-									<td>k<sub>i</sub></td>
-									<td>Association rate for derepression mechanism by IPTG</td>
-									<td>3 x 10<sup>-5</sup></td>
-									<td>nM<sup>-1</sup>min<sup>-1</sup></td>
-									<td></td>
-								</tr>
-								<tr>
-									<td>k<sub>-i</sub></td>
-									<td>Dissociation rate for derepression mechanism</td>
-									<td>4.8 x 10<sup>3</sup></td>
-									<td>min<sup>-1</sup></td>
-									<td></td>
-								</tr>
-								<tr>
-									<td></td>
-									<td>Transcription rate</td>
-									<td>2</td>
-									<td>mRNA.min<sup>-1</sup>nM<sup>-1</sup></td>
-									<td></td>
-								</tr>
-								<tr>
-									<td>k<sub>r</sub></td>
-									<td>Association rate of ribosome with r.b.s</td>
-									<td>1</td>
-									<td>min<sup>-1</sup>mRNA<sup>-1</sup></td>
-									<td></td>
-								</tr>
-								<tr>
-									<td>k<sub>-r</sub></td>
-									<td>Dissociation rate of ribosome with r.b.s</td>
-									<td>1</td>
-									<td>min<sup>-1</sup></td>
-									<td></td>
-								</tr>
-								<tr>
-									<td></td>
-									<td>Translation rate</td>
-									<td>4</td>
-									<td>nM.min<sup>-1</sup>mRNA<sup>-1</sup></td>
-									<td></td>
-								</tr>
-								<tr>
-									<td>k<sub>1</sub></td>
-									<td>Association rate of TEV with its substrate in the cytoplasm</td>
-									<td>7.8 x 10<sup>-7</sup></td>
-									<td></td>
-									<td></td>
-								</tr>
-								<tr>
-									<td>k<sub>-1</sub></td>
-									<td>Dissociation rate of TEV with its substrate in the cytoplasm</td>
-									<td>6 x 10<sup>-4</sup></td>
-									<td></td>
-									<td></td>
-								</tr>
-								<tr>
-									<td>k<sub>2</sub></td>
-									<td>Cleaving rate by TEV in cytoplasm</td>
-									<td>1.38 x 10<sup>-2</sup></td>
-									<td></td>
-									<td></td>
-								</tr>
-								<tr>
-									<td>k<sub>3</sub></td>
-									<td>Association rate of NGF and TEV with transmembrane transporter</td>
-									<td>6 x 10<sup>-4</sup></td>
-									<td></td>
-									<td></td>
-								</tr>
-								<tr>
-									<td>k<sub>-3</sub></td>
-									<td>Dissociation rate of NGF and TEV with transporter</td>
-									<td>2.34</td>
-									<td></td>
-									<td></td>
-								</tr>
-								<tr>
-									<td>k<sub>4</sub></td>
-									<td>Translocation rate within the transporter</td>
-									<td>2.1</td>
-									<td></td>
-									<td></td>
-								</tr>
-								<tr>
-									<td>k<sub>f</sub></td>
-									<td>NGF folding rate in the medium</td>
-									<td>0.28</td>
-									<td></td>
-									<td></td>
-								</tr>
-								<tr>
-									<td>k<sub>5</sub></td>
-									<td>Association rate of TEV with its substrate in the medium</td>
-									<td>7.8 x 10<sup>-5</sup></td>
-									<td></td>
-									<td></td>
-								</tr>
-								<tr>
-									<td>k<sub>-5</sub></td>
-									<td>Dissociation rate of TEV with its substrate in the medium</td>
-									<td>0.06</td>
-									<td></td>
-									<td></td>
-								</tr>
-								<tr>
-									<td>k<sub>6</sub></td>
-									<td>Cleaving rate by TEV in the medium</td>
-									<td>1.38</td>
-									<td></td>
-									<td></td>
-								</tr>
-							</table>
-						</div>
-						<div class="block separator"></div>
-						<div>
-							<h3>MODEL RESULTS</h3>
-						</div>
-						<div class="block separator"></div>
-					</div>
-				</div>
-				<div class="block separator"></div>
-			<!-- Second Onglet Diffusion of NGF -->
+            <div class="block full">
-				<div class="block full bothContent">
+                <p>Alignment of <b>Sequencing</b> Results then confirmed that pSB1C3 contained Seq1 and Seq2, <b><a href="">BBa_K2616000 </a></b>. </p>
-					<div class="block dropDown" id="Diffusion">
+            </div>
-						<h4>NGF diffusion simultation in a given environment</h4>
+            <div class="block two-third center">
-					</div>
+                <img src="https://static.igem.org/mediawiki/2018/e/ee/T--Pasteur_Paris--Sequencing_proNGF.PNG">
+                <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>
+            </div>
+            <div class="block full">
+                 <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>
+            </div>
-					<div class="block hiddenContent">
+        <div class="block full">
-						<span class="closeCross"><img src="https://static.igem.org/mediawiki/2018/6/67/T--Pasteur_Paris--CloseCross.svg"></span>
+                <h4 style="text-align: left;">proNGF characterization and purification</h4><br><br>
-						<div class="block title">
-							<h1 style="padding-top: 50px;">NGF diffusion diffusion in a given environment</h1><br>
-							<p><i>We are looking to understand the way the NGF spreads inside the conduit once it is produced. This will help us to determine the NGF concentration u(x,t) (ng.mL<SUP>-1</SUP>) as a function of the distance x (cm) from the production site of NGF.</i></p>
-						</div>
-						<!-- Fick's diffusion law -->
-							<div class="block full">
-								<h3>Fick’s diffusion law </h3>
-								<p>To simulate NGF diffusion in the microfluidic chip we consider a unidimensional conduit of axe x and a constant concentration of NGF introduced at one end of the canals. In this part, diffusion is assumed to be the only mechanism producing the gradient decay in the micro canals. We can model the diffusion characteristics of NGF with Fick’s second law of diffusion:<br>
-								<span style="position: relative; display: inline-block; width: 100%; text-align: center;">
-									<span class="frac">
-										<span>du</span>
-										<span class="symbol">/</span>
-										<span class="bottom">dt</span>
-									</span>
-									 = C<SUB>diff</SUB>
-									<span class="frac">
-										<span>d<SUP>2</SUP>u</span>
-										<span class="symbol">/</span>
-										<span class="bottom">dx<SUP>2</SUP></span>
-									</span>
-									&emsp;&emsp;&emsp;(1)
-								</span>
-								</p>
-								<p>Cdiff is assumed to be constant inside the conduit and depends on the material used.<br>
-								There are also two boundary conditions:<br>
-								at x=0: &emsp;&emsp;
-									<span class="frac">
-										<span>du</span>
-										<span class="symbol">/</span>
-										<span class="bottom">dx</span>
-									</span>
-									 |<SUB>(0,t)</SUB>&emsp;&emsp;&emsp;(2)<br>
-								at x=L: &emsp;&emsp;
-								<span style="text-align: left;">
-									<span class="frac">
-										<span>du</span>
-										<span class="symbol">/</span>
-										<span class="bottom">dx</span>
-									</span>
-									 |<SUB>(L,t)</SUB>&emsp;&emsp;&emsp;(3)<br>
-								</span>
-								</p>
-								<p>Indeed, in the same material, the rate transfer of the diffusing NGF through the cross section of the micro canal is proportional to the concentration gradient normal to the cross section. It is assumed that the leakage of NGF at both ends of the micro canal is negligible because there should be little NGF at the ends the micro canals compared to the total amount of NGF and second because of a low NGF diffusion rate.
-								The equation (1) can be solved with Euler’s method and we find the NGF concentration gradient at the position x and time t. The MatLab code is the following:</p>
-							</div>
-							<div class="block half">
-								<img src="https://static.igem.org/mediawiki/2018/6/66/T--Pasteur_Paris--code.1.svg">
-							</div>
-							<div class="block half">
-								<p>We displayed our results showing a decrease of the concentration of NGF (u(x,t)) depending on the distance of the conduit x.</p>
-								<img src="https://static.igem.org/mediawiki/2018/f/f3/T--Pasteur_Paris--code-plot.1.svg">
-							</div>
-							<div class="block half">
-								<p>We used the following parameters for the model: </p>
-								<table class="tableData">
-									<tr>
-										<td>Length of the conduit: L</td>
-										<td>0.1 cm </td>
-									</tr>
-									<tr>
-										<td>Diffusion coefficient of NGF : Cdiff</td>
-										<td>7,8*10<SUP>-7</SUP> cm<SUP>2</SUP>.s<SUP>-1</SUP></td>
-									</tr>
-									<tr>
-										<td>Time length of the experiment: t_final</td>
-										<td>3 600 s </td>
-									</tr>
-								</table>
-							</div>
-							<div class="block full">
-								<p style="text-align: center;">We obtain the following graphs: </p>
-							</div>
-							<div class="block half">
+    <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 <b><a href="">BBa_K2616000 </a></b> 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>
-								<img src="https://static.igem.org/mediawiki/2018/b/b4/T--Pasteur_Paris--gif.gif" style="max-width: 500px; box-shadow: 0px 0px 8px -2px;">
+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>
-							</div>
+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>
-							<div class="block half">
-								<img src="https://static.igem.org/mediawiki/2018/1/14/T--Pasteur_Paris--gifcouleurspuce.gif" style="max-width: 500px; box-shadow: 0px 0px 8px -2px;">
-							</div>
-						<!-- Optimisation of the gradient -->
-							<div class="block full">
-								<h3>Optimisation of the NGF gradient</h3>
-							</div>
-							<div class="block half">
-								<p>To optimize the accuracy of the NGF gradient we interpolate the curve u(x)=f(x). Consequently, we obtain the f polynomial function easier to derive and a polynomial function of the gradient with a better accuracy than with the first method. The program is the following:</p>
-							</div>
-							<div class="block half">
-								<img src="https://static.igem.org/mediawiki/2018/2/2f/T--Pasteur_Paris--interpolation-plot.1.svg">
-							</div>
-							<div class="block two-third">
-							</div>
-							<div class="block two-third">
-								<p>With the same parameters as with the previous model we obtain the following graphs: </p>
-							</div>
-							<div class="block two-third">
-							</div>
-						<!-- Analysis of the model -->
-							<div class="block full">
-								<h3>Analysis of the model </h3>
-								<p><i>To validate the model, we vary the three parameters (L, t_final, C<SUB>diff</SUB>) to verify if the program corresponds to a diffusion phenomenon described in Fick’s second law of diffusion. </i></p>
-							</div>
-							<div class="block two-third">
-								<img src="https://static.igem.org/mediawiki/2018/4/41/T--Pasteur_Paris--gif-fct-L.gif" style="max-width: 500px; box-shadow: 0px 0px 8px -2px;">
-							</div>
-							<div class="block full">
-								<p>Observations:<br>
-									<ol style="text-align: left; list-style-type: disc;">
-										<li>When the length of the conduit increases but the duration of the experiment is fixed the NGF doesn’t have the time to diffuse in the entire conduit.</li>
-										<li>For instance, with a t_final= 3 600s the NGF molecules can’t diffuse further than x=0.2cm.</li>
-									</ol>
-								</p>
-							</div>
-							<div class="block two-third">
-								<img src="https://static.igem.org/mediawiki/2018/0/07/T--Pasteur_Paris--fct-Cdiff.gif" style="max-width: 500px; box-shadow: 0px 0px 8px -2px;">
-							</div>
-							<div class="block full">
-								<p>The higher the diffusion coefficient, the faster the molecules will diffuse in the conduit. Indeed, we observe in the model that with a fixed t_final:<br>
-									<ol style="text-align: left; list-style-type: disc;">
-										<li>NGF concentration at x=0.1 cm is 675 000 ng.ml<SUP>-1</SUP> for a diffusion coefficient C<SUB>diff</SUB> = 15*10<SUP>-7</SUP> cm<SUP>2</SUP>.s<SUP>-1</SUP></li>
-										<li>For a diffusion coefficient two times lower, the NGF concentration is 380 ng.ml<SUP>1</SUP></li>
-									</ol>
-								</p>
-								<p>The results confirm the prediction of the Fick’s law model. </p>
-							</div>
-							<div class="block full">
-								<p>When the time length of the experiment lasts from 1 hour to 2 hours, the concentration of NGF is almost homogeneous in the entire conduit. At the end of the conduit, for x= 0.1 cm, the concentration of NGF equals to 910 ng.ml-1 when t_final= 7 200s whereas the concentration is 3 900 ng.ml<SUP>-1</SUP> when t_final=3 600s. </p>
-								<p>It is interesting to observe that when the duration of the experiment increases, the stationary regime is established: the NGF concentration in the conduit becomes independent of the position and time. Indeed, the concentation gradient of NGF in the conduit moves toward 0 for any position. </p>
-							</div>
-							<div class="block two-third">
-							</div>
-							<div class="block two-third">
-							</div>
-					</div>
-				</div>
-				<div class="block separator"></div>
-			<!-- Third Onglet Neurons Growth-->
-				<div class="block full bothContent">
-					<div class="block dropDown" id="Growth">
-						<h4>Neurons growth in the presence of NGF</h4>
-					</div>
-					<div class="block hiddenContent">
+            </div>
-						<span class="closeCross"><img src="https://static.igem.org/mediawiki/2018/6/67/T--Pasteur_Paris--CloseCross.svg"></span>
+            <div class="block two-third center">
-						<div class="block title">
+                <img src="https://static.igem.org/mediawiki/2018/6/69/T--Pasteur_Paris--ResultsFPLC.png">
-							<h1>Neurons growth in the presence of NGF</h1><br>
+                <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>
-							<p><i>In this part our goal is to determine the length of the neurite outgrowth (g(t)) in response to the gradient concentration of NGF.</i></p>
+            </div>
-						</div>
+            <div class="block full">
-						<!-- Explanation of the model -->
+                <p>We analyzed bacterial lysate and purification fractions using SDS-PAGE electrophoresis and Mass spectrometry. </p>
-							<div class="block full">
+            </div>
-								<h3>Explanation of the model</h3>
+            <div class="block half">
-							</div>
+                <img src="https://static.igem.org/mediawiki/2018/5/53/T--Pasteur_Paris--SDSPage.png">
-							<div class="block full">
-								<h5 style="text-align: left">Baseline growth rate: </h5>
-								<p>In our mathematical model, neurites grow at a constant growth rate defined as the baseline growth rate G0  when the concentration is below the threshold (assumed to be 995 ng.mL<SUP>-1</SUP>). Neurites stop growing when the NGF concentration is higher than the threshold concentration. The value for the baseline growth rate G0 has been fixed at 20 <FONT face="Raleway">&mu;</FONT>m.h<SUP>-1</SUP> for this model. </p>
-								<h5 style="text-align: left">Concentration Gradient:</h5>
-								<p>The extent of directional guidance is gradient steepness-dependent provided that the concentration gradient reaches the threshold value. The gradient factor k is a gradient steepness-dependent positive effect on the neurite growth rate. </p>
-								<p>In this model we assume that the baseline growth rate and the growth rate in the presence of concentration gradient follow an additive rule. This can be explained by the fact that both the NGF concentration and the its gradient can both individually contribute to neurite extension. The equation governing neurite outgrowth thus becomes:<br><br>
-								<span style="position: relative; display: inline-block; text-align: center; width: 100%">
-									<span class="frac">
-										<span>dg</span>
-										<span class="symbol">/</span>
-										<span class="bottom">dt</span>
-									</span>
-									 = G<SUB>0</SUB> + k
-									<span class="frac">
-										<span><FONT face="Raleway">&part;</FONT>u</span>
-										<span class="symbol">/</span>
-										<span class="bottom"><FONT face="Raleway">&part;</FONT>x</span>
-									</span>
-									 |<SUB>(g(t),t)</SUB> &emsp; &emsp; &emsp; (4)</p>
-								</span>
-							</div>
-							<div class="block full">
-								<p>We can introduce a time parameter Tlag because the time taken to transmit the NGF signal is finite. The experiments show that the time lag for the cells to respond to NGF is approximately 1 day. The experiments show:<br>
-								if t <FONT face="Raleway">&le;</FONT> T<SUB>lag</SUB> : &emsp; &emsp;
-									<span class="frac">
-										<span>dg</span>
-										<span class="symbol">/</span>
-										<span class="bottom">dt</span>
-									</span> = 0<br>
-								else, if: t <FONT face="Raleway">&ge;</FONT> T<SUB>lag</SUB> : &emsp; &emsp;
-									<span class="frac">
-										<span>dg</span>
-										<span class="symbol">/</span>
-										<span class="bottom">dt</span>
-									</span> = G<SUB>0</SUB> + k
-									<span class="frac">
-										<span><FONT face="Raleway">&part;</FONT>u</span>
-										<span class="symbol">/</span>
-										<span class="bottom"><FONT face="Raleway">&part;</FONT>x</span>
-									</span>
-								 |<SUB>(g(t),t)</SUB>
-								</p>
-							</div>
-						<!-- Solving the model -->
-							<div class="block full">
-								<h3>Solving the model</h3>
-							</div>
-							<div class="block full">
-								<p>To solve the equation (4) we are using Euler’s method forward because the gradient concentration of NGF depends on the length of the neurite (since neurites consume NGF). <br><br>
-								The Equation (4): &emsp; &emsp; <br>
-								<span style="position: relative; display: inline-block; width: 100%; text-align: center;">
-									<span class="frac">
-										<span>dg</span>
-										<span class="symbol">/</span>
-										<span class="bottom">dt</span>
-									</span> = G<SUB>0</SUB> + k
-									<span class="frac">
-										<span><FONT face="Raleway">&part;</FONT>u</span>
-										<span class="symbol">/</span>
-										<span class="bottom"><FONT face="Raleway">&part;</FONT>x</span>
-									</span>
-								 |<SUB>(g(t),t)</SUB>
-								</span><br><br>
-								Can be written as: &emsp; &emsp; <br><br>
-								<span style="position: relative; display: inline-block; width: 100%; text-align: center;">
-									g' = G<SUB>0</SUB> + k*f(g,t)
-								</span><br><br>
-								Which can be written as : &emsp; &emsp; <br><br>
-								<span style="position: relative; display: inline-block; width: 100%; text-align: center;">
-									<span class="frac">
-										<span>g<SUB>n+1</SUB> - g<SUB>n</SUB></span>
-										<span class="symbol">/</span>
-										<span class="bottom">dt</span>
-									</span>
-									 |<SUB>(g(t),t)</SUB> = G<SUB><FONT face= "Raleway">&theta;</FONT></SUB> + k
-									<span class="frac">
-										<span><FONT face="Raleway">&part;</FONT>u</span>
-										<span class="symbol">/</span>
-										<span class="bottom"><FONT face="Raleway">&part;</FONT>x</span>
-									</span>
-									 |<SUB>(g(t),t)</SUB>
-								</span><br><br>
-								We can therefore have an expression of g<SUB>n+1</SUB>: &emsp; &emsp; <br><br>
-								<span style="position: relative; display: inline-block; width: 100%; text-align: center;">
-									g<SUB>n+1</SUB> = g<SUB>n</SUB> + dt*[G<SUB>0</SUB> + k
-									<span class="frac">
-										<span><FONT face="Raleway">&part;</FONT>u</span>
-										<span class="symbol">/</span>
-										<span class="bottom"><FONT face="Raleway">&part;</FONT>x</span>
-									</span>
-									 |<SUB>(g(t),t)</SUB>]
-								</span><br><br>
-								With initial values of g<SUB><FONT face="Raleway">&theta;</FONT></SUB>, t<SUB><FONT face="Raleway">&theta;</FONT></SUB> and
-									<span class="frac">
-										<span><FONT face="Raleway">&part;</FONT>u</span>
-										<span class="symbol">/</span>
-										<span class="bottom"><FONT face="Raleway">&part;</FONT>x</span>
-									</span>
-									 |<SUB>(g(t),t)</SUB>
-								  we can find all the values of the g </p>
-							</div>
-					</div>
+                <div class="legend"><b>Figure 6: </b>SDS-PAGE gel Bis-Tris 4-12% of bacterial lysate and proNGF purification fraction by SDS-PAGE.
-				</div>
+ </div>
-				<div class="block separator"></div>
+            </div>
+            <div class="block half">
+<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>
+</div>
+<div class="block half">
+<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>
+</div>
-			<div class="block title">
+            <div class="block half">
-				<h1 id="References">References</h1>
+                <img src="https://static.igem.org/mediawiki/2018/c/cd/T--Pasteur_Paris--distribution.png">
-			</div>
+                <div class="legend"><b>Figure 7: </b>Distribution of proNGF and TEV protease by gel fractions after mass spectrometry analysis. </div>
-			<div class="block full">
+            </div>
-				<p>[1] Defining the concentration gradient of nerve growth factor for guided neurite outgrowth, XCao M.SShoichet, March 2001</p>
-				<p>[2] Immobilized Concentration Gradients of Neurotrophic Factors Guide Neurite Outgrowth of Primary Neurons in Macroporous Scaffolds, Moore K, MacSween M, Shoichet M, feb 2006</p>
-				<p>[3] Mathematical Modeling of Guided Neurite Extension in an Engineered Conduit with Multiple Concentration Gradients of Nerve Growth Factor (NGF), Tse TH, Chan BP, Chan CM, Lam J, sep 2007</p>
-				<p>[4] Mathematical modelling of multispecies biofilms for wastewater treatment, Maria Rosaria Mattei, november 2005</p>
-			</div>
-		</div>
-	</div>
+            <div class="block full">
+<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>
+</div>
+            <div class="block two-third center">
+                <img src="https://static.igem.org/mediawiki/2018/b/ba/T--Pasteur_Paris--Align_sequence_Mass.png">
+                <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>
+            </div>
+            <div class="block two-third center">
+                <img src="https://static.igem.org/mediawiki/2018/2/20/T--Pasteur_Paris--massspec.png">
+                <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>
+            </div>
+            <div class="block full">
+                <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>
+<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.
+</p></div>
+            <div class="block two-third center">
+                <img src="https://static.igem.org/mediawiki/2018/5/56/T--Pasteur_Paris--WBproNGF.png">
+                <div class="legend"><b>Figure 10: </b>Western Blot analysis of batch purification proNGF under native and partial denaturing conditions. </div>
+            </div>
+            <div class="block separator-mark"></div>
+                    </div>
+         <div class="block full" style="background-color: transparent;">
+                <p><i>Achievements: </i><br>
+                    <ul style="text-align: left;">
+                        <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=BBa_K2616000 "http://parts.igem.org/Part:BBa_K2616000"> BBa_K2616000</a> </li>
+                        <li>Successfully sequenced <a href=BBa_K2616000 "http://parts.igem.org/Part:BBa_K2616000"> BBa_K2616000</a> in pSB1C3 and sent to iGEM registry </li>
+                        <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>
+                        <li>Successfully characterized production of NGF thanks to mass spectrometry</li>
+                        <li>Successfully <b>observe axon growth</b> in microfluidic chip in presence of commercial NGF</li>
+                    </ul><br></p>
+                    <p><i>Next steps:</i><br>
+                    <ul style="text-align: left;">
+                        <li><b>Purify</b> secreted NGF, and characterize its effects on neuron growth thanks to our microfluidic device </li>
+                        <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>
+                    </ul>
+                </p>
+                    </div>
+                    </div>
+<!-- Second Onglet Fight infections-->
+                <div class="block full bothContent">
+                    <div class="block dropDown" id="Fight">
+                                       <h4>FIGHT INFECTIONS : Click to see more</h4>
+                        </div>
+                    <div class="block hiddenContent">
+                        <span class="closeCross"><img src="https://static.igem.org/mediawiki/2018/6/67/T--Pasteur_Paris--CloseCross.svg"></span>
+            <div class="block title" id="Fight">
+                <h1>FIGHT INFECTIONS</h1><br>
+            </div>
+            <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>
+</div>
+<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 vectors contained the insert by electrophoresis (Figure 12,13).<br>
+</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>
+        <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/2018/7/77/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>
+            <div class="block separator-mark"></div>
+                    </div>
+         <div class="block full" style="background-color: transparent;">
+                <i><p>Achievements:<br></i>
+                    <ul style="text-align: left;">
+                        <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><i>Next steps:<br></i>
+                    <ul style="text-align: left;">
+                        <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>
+      </div>
+      </div>
+<!-- Third Onglet Kill switch-->
+                <div class="block full bothContent">
+                    <div class="block dropDown" id="Kill">
+                        <h4>KILL SWITCH: Click to see more </h4>
+                    </div>
+                    <div class="block hiddenContent">
+                        <span class="closeCross"><img src="https://static.igem.org/mediawiki/2018/6/67/T--Pasteur_Paris--CloseCross.svg"></span>
+                        <div class="block title">
+                            <h1 style="padding-top: 50px;">KILL SWITCH</h1>
+      </div>
+      <div class="block half">
+        <p>Once we received the sequences encoding for this production cassette (named construction Seq9) in commercial plasmids, in order to have more DNA, we transformed competent bacteria <i>E. coli</i> DH5alpha resulting in clones. After bacteria culture and plasmid DNA extraction, we digested commercial vectors with restriction enzymes, extracted the inserts from the gel, and ligated it into linearized pSB1C3 for iGEM submission and expression in BL21(DE)3.</p>
+                <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 contained the insert by electrophoresis.</p>
+      </div>
+            <div class="block half">
+        <img src="https://static.igem.org/mediawiki/2018/c/c5/T--Pasteur_Paris--GelKS.png">
+        <div class="legend"><b>Figure 7: </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"style="font-weight: bold ; color:#85196a;"target="_blank"> 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>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>
+            </div>
+            <div class="block full">
+        <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 22: </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>
+      </div>
+            <div class="block separator-mark"></div>
+    </div>
+         <div class="block full" style="background-color: transparent;">
+<p><i><p>Achievements:<br></i>
+          <ul style="text-align: left;" style="text-align: left;">
+            <li>Successfully cloned a part coding for toxin/antitoxin (CcdB/CcdA) system in  iGEM plasmid backbone, creating a <b>new composite part</b></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><i>Next steps:</i><br>
+          <ul style="text-align: left;">
+            <li>Find a system that kills bacteria when released in the environment rather than just stopping their growth</li>
+          </ul>
+        </p></p></div>
+    </div>
+ <!-- Fourth Onglet Membrane-->
+                <div class="block full bothContent">
+                    <div class="block dropDown" id="Membrane">
+                        <h4>MEMBRANE: Click to see more</h4>
+                    </div>
+                    <div class="block hiddenContent">
+                        <span class="closeCross"><img src="https://static.igem.org/mediawiki/2018/6/67/T--Pasteur_Paris--CloseCross.svg"></span>
+                        <div class="block title" id ="Membrane">
+                            <h1 style="padding-top: 50px;">Membrane</h1>
+            </div>
+                    <div class="block two-third">
+                         <p>The membrane filter is a key element of our prosthesis system, allowing the confinement of the genetically modified bacteria and the conduction of neuron impulses. We tested two types of membranes: Sterlitech Polycarbonate Gold-Coated Membrane Filters (pores diameter of 0.4 micrometer) and Sterlitech Alumina Oxide Membrane Filters (pores diameter of 0.2 micrometer).<br>
+                         Sterlitech Alumina Oxide Membrane Filters were coated with different types of biocompatible conductive polymers: PEDOT:PSS (poly(3,4-ethylenedioxythiophene) polystyrene sulfonate), PEDOT:Cl and PEDOT:Ts.<br>
+                         To characterize the potential of the different types of membranes to be integrated in our prosthesis system, we evaluated the coating of the alumina oxide membranes, their biocompatibility and their electrical conductivity.<br></p>
+       </div>
+                    <div class="block one-third">
+        <img src="https://static.igem.org/mediawiki/2018/9/9c/T--Pasteur_Paris--PEDOT-PSS.jpg">
+        <div class="legend"><b>Figure 23: </b> PEDOT:PSS </div>
+       </div>
+                    <div class="block full">
+        <h2 style="text-align: left;">Biocompatibility</h2>
+        <p></p>
+       </div>
+                    <div class="block full">
+        <h2 style="text-align: left;">Conductivity</h2>
+        <p></p>
+       </div>
+                    <div class="block one-third">
+        <img src="https://static.igem.org/mediawiki/2018/8/88/T--Pasteur_Paris--Well-chip.jpg" >
+        <div class="legend"><b>Figure 24: </b> Hand-made PDMS well chip </div>
+        </div>
+                    <div class="block two-third">
+        <p>The conductivity of the membranes was measured on a self-made device. It consists of a culture well made of PDMS (polydimethylsiloxane), with a membrane filter at its bottom and a platinum wire linking the conductive membrane filter with the exterior.</p>
+       </div>
+                    <div class ="block full">
+        <h3 style="text-align: left;">Platinum wire</h3>
+        <p>The voltage difference between the two extremities of the wire was measured. </p>
+       </div>
+                    <div class="block two-third center">
+        <img src="https://static.igem.org/mediawiki/2018/5/51/T--Pasteur_Paris--Wire-conductivity.PNG">
+        <div class="legend"><b>Figure 25: </b> Voltage difference between the two extremities of the platinum wire.</div>
+       </div>
+                    <div class="block full">
+        <p>The voltage difference between different platinum wires is pretty much the same. As we want to compare the differences between multiple membranes, we don't need to take into account the variability from one chip to another of the platinum wire's resistance. That means, it is meaningful to measure the voltage difference between a point on the membrane and the extremity of the wire outside the well, and use this data to compare the membranes. </p>
+       </div>
+                    <div class="block full">
+        <h3 style="text-align: left;">Membranes</h3>
+        <p>The voltage difference between a point on the membrane  (located near the border of the membrane filter) and the extremity of the platinum wire outside the well was measured.</p>
+       </div>
+                    <div class="block two-third center">
+        <img src="https://static.igem.org/mediawiki/2018/2/2e/T--Pasteur_Paris--Membrane-conductivity.PNG">
+        <div class="legend"><b>Figure 26: </b> Voltage difference between the extremity of the platinum wire outside the well and a point on the membrane.</div>
+       </div>
+      </div>
+    </div>
+  </div>
 </html>

Difference between revisions of "Team:Pasteur Paris/TrainingThomas2"

Revision as of 17:38, 16 October 2018

RECONNECT NERVES: Click to see more

RECONNECT NERVES

DNA assembly

proNGF characterization and purification

FIGHT INFECTIONS : Click to see more

FIGHT INFECTIONS

RIP Secretion BBa_K2616001

Fluorescence reading experiments

Crystal violet staining

Biofilm PFA fixation before staining

S. aureus Detection and RIP secretion BBa_K2616003

KILL SWITCH: Click to see more

KILL SWITCH

MEMBRANE: Click to see more

Membrane

Biocompatibility

Conductivity

Platinum wire

Membranes