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<h4>MEMBRANE<br> <i>Click to see more </i></h4> | <h4>MEMBRANE<br> <i>Click to see more </i></h4> | ||
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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 ="Membrane"> | <div class="block title" id ="Membrane"> | ||
<h1 style="padding-top: 50px;">Membrane</h1> | <h1 style="padding-top: 50px;">Membrane</h1> | ||
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<h2 style="text-align: left;">Conductivity</h2> | <h2 style="text-align: left;">Conductivity</h2> | ||
− | <p> The membranes used in our system should possess good electric conductive capabilities for nerve influx conduction. The goal here is to evaluate the conductivity of the membranes | + | <p> The membranes used in our system should possess good electric conductive capabilities for nerve influx conduction. The goal here is to evaluate the conductivity of the membranes.</p> |
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<p>As in the end we are going to measure the conductivity of the system biofilm+membrane+platinum wire, we want to simplify the measurements and neglect the impact of the platinum wire. Function generator was set on sine. The physical quantities measured here are Eg, the generator's tension amplitude and Ep, the voltage difference between the two extremities of a platinum wire. the quantity calculated here is 20*log(Ep/Eg) for different frequencies. </p> | <p>As in the end we are going to measure the conductivity of the system biofilm+membrane+platinum wire, we want to simplify the measurements and neglect the impact of the platinum wire. Function generator was set on sine. The physical quantities measured here are Eg, the generator's tension amplitude and Ep, the voltage difference between the two extremities of a platinum wire. the quantity calculated here is 20*log(Ep/Eg) for different frequencies. </p> | ||
<h4 style="text-align: left;"> Results </h4> | <h4 style="text-align: left;"> Results </h4> | ||
− | <img src="https://static.igem.org/mediawiki/2018/c/c4/T--Pasteur_Paris--Conductivity-platinum-wire.jpg" > | + | <img src="https://static.igem.org/mediawiki/2018/c/c4/T--Pasteur_Paris--Conductivity-platinum-wire.jpg" style="width:500px"> |
<div class="legend"><b>Figure 24: </b> Conductivity of a platinum wire for different frequencies </div> | <div class="legend"><b>Figure 24: </b> Conductivity of a platinum wire for different frequencies </div> | ||
<h4 style="text-align: left;"> Interpretation </h4> | <h4 style="text-align: left;"> Interpretation </h4> | ||
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<h3>Frequency impact on membrane conductivity</h3> | <h3>Frequency impact on membrane conductivity</h3> | ||
− | <p> Before measuring the conductivity of multiple membranes, we need to have an overview of the impact of the frequency on the | + | <p> Before measuring the conductivity of multiple membranes, we need to have an overview of the impact of the frequency on the conductivity of a membrane. We tested two gold-coated membranes.Function generator was set on sine. The physical quantities measured here are Eg, the generator's tension amplitude and Ep, the voltage difference between the extremity of the platinum wire outside the well chip and a point on the edge of the membrane of the chip. the quantity calculated here is 20*log(Ep/Eg) for different frequencies. </p> |
<h4 style="text-align: left;"> Results </h4> | <h4 style="text-align: left;"> Results </h4> | ||
− | <img src="https://static.igem.org/mediawiki/2018/ | + | <img src="https://static.igem.org/mediawiki/2018/6/6e/T--Pasteur_Paris--Gold-membrane-well-chip-conductivity.jpg" style="width:500px"> |
<div class="legend"><b>Figure 24: </b> Conductivity of a platinum wire for different frequencies </div> | <div class="legend"><b>Figure 24: </b> Conductivity of a platinum wire for different frequencies </div> | ||
<h4 style="text-align: left;"> Interpretation </h4> | <h4 style="text-align: left;"> Interpretation </h4> | ||
− | <p> Voltage difference calculated is | + | <p> Voltage difference calculated is very low, indicating a very good conductivity for the gold-coated membrane. Technically, we measured the conductivity of the system membrane+platinum wire, but we showed that the wire's conductivity could be neglected. Resistance increases in higher frequencies, again because of the skin-effect in metals. But as we are going to use only low frequencies, this doesn't affect us, and moreover, the frequency response is flat for wide range of low frequencies. </p> |
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− | <img src="https://static.igem.org/mediawiki/2018/ | + | <h3>Membrane conductivity</h3> |
− | <div class="legend"><b>Figure | + | <p> We measured the conductivity of 6 membranes on PDMS well chips (2 gold-coated, 1 bare alumina oxide, 1 PEDOT:PSS-coated, 1 PEDOT:Cl-coated, 1 PEDOT:Ts-coated). Here we show the electric circuit that we used for the following experiments. </p> |
− | </div> | + | <img src="https://static.igem.org/mediawiki/2018/b/bb/T--Pasteur_Paris--Electrical_circuit_1.pdf" style="width:500px"> |
+ | <p> Function generator was set on square at 200 Hz. The physical quantities measured are Eg, the generator tension amplitude, and Ep, the amplitude of the voltage difference between a point on the membrane inside the well and the extremity of the platinium strip outside the well. Tension amplitude of the resistor is given by Er = Eg - Ep. Current flowing through the electric circuit is calculated with I = Er/R. Conductivity of the membrane is given by I/Ep. Conductivity of each membrane was measured 3 times. Figure 1 shows the mean value for each membrane and the standard deviation. </p> | ||
+ | <h4 style="text-align: left;"> Results </h4> | ||
+ | <img src="https://static.igem.org/mediawiki/2018/5/50/T--Pasteur_Paris--Membrane-Conductivity.jpg" style="width:500px"> | ||
+ | <div class="legend"><b>Figure 24: </b> Conductivity of a platinum wire for different frequencies </div> | ||
+ | <h4 style="text-align: left;"> Interpretation </h4> | ||
+ | <p> Bare alumina oxide and PEDOT:PSS-coated membranes show similar conductivies, indicating the incomplete coating of PEDOT:PSS on alumina oxide membranes. On the opposite, PEDOT:Cl and PEDOT:Ts seem to exhibit on average better conductivities, but in the same time, the coating of these membranes revealed by electron microscopy seemed to have covered the alumina oxide membranes in a more uniform way, ensuring enhanced conductive capabilities. These results can be criticized because of the high deviation and because the membranes conductivity was measured after several biofilms were grown on them, which may have affected the measurements. </p> | ||
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+ | <h2 style="text-align: left;">Biocompatibility and biofilm conductivity</h2> | ||
+ | <p> One last important property of the membranes to measure is the capability of bacteria to form a biofilm on them, as in our prosthesis system, the membrane is going to be directly in contact with the genetically modified biofilm, as well as the human body. </p> | ||
+ | <p> We used the last section of the following protocol to form biofilms in our PDMS well chips and to measure the biofilm growth: <a href="https://static.igem.org/mediawiki/2018/b/b5/T--Pasteur_Paris--PDMS-well-chip.pdf"> click here </a> </p> | ||
+ | <h4 style="text-align: left;"> Results: biofilm growth </h4> | ||
+ | <p> Biofilm growth was measured 4 times in total. For each series of measurements, the measured optical densities were divided by the optical density of the base liquid culture, to normalize the measures.</p> | ||
+ | <img src="https://static.igem.org/mediawiki/2018/8/84/T--Pasteur_Paris--Biofilm-Growth.jpg" style="width:500px"> | ||
+ | <div class="legend"><b>Figure 24: </b> Biofilm growth (mean value and standard deviation for each type of membrane)</div> | ||
+ | <h4 style="text-align: left;"> Results: biofilm conductivity </h4> | ||
+ | <p> For conductivity measurements, we used the same electric circuit as in figure... . Function generator was set on square at 200 Hz. The physical quantities measured are Eg, the generator tension amplitude, and Ep, the amplitude of the voltage difference between a point on the biofilm inside the well and the extremity of the platinium strip outside the well. Tension amplitude of the resistor is given by Er = Eg - Ep. Current flowing through the electric circuit is calculated with I = Er/R. Conductivity of the membrane is given by I/Ep. Conductivity of each membrane with a biofilm was repeated 3 times. </p> | ||
+ | <img src="https://static.igem.org/mediawiki/2018/8/83/T--Pasteur_Paris--Conductivity-with-biofilm.jpg" style="width:500px"> | ||
+ | <div class="legend"><b>Figure 24: </b> Membrane conductivity with biofilm (mean value and standard deviation for each type of membrane)</div> | ||
+ | <p> To approximate very roughly the conductivity of the biofilm, the average conductivity values of the membranes with a biofilm were divided by the corresponding average biofilm growth values, and the conductivity of the membrane was then substracted. </p> | ||
+ | <img src="https://static.igem.org/mediawiki/2018/6/61/T--Pasteur_Paris--Biofilm-Conductivity.jpg" style="width:500px"> | ||
+ | <div class="legend"><b>Figure 24: </b>Estimated biofilm conductivity </div> | ||
+ | <h4 style="text-align: left;"> Interpretation </h4> | ||
+ | <p> As told by the membrane manufacturer, biofilm formation on gold membranes seems indeed to be more difficult than on other membranes. However we expected PEDOT:PSS-coated membranes to stimulate more the growth of biofilm, but perhaps that may be just another indicator of the incomplete coating. Surprisingly, PEDOT:Cl tends to allow better formation of biofilms. We realized only after the experiments the need for a control biofilm culture without membrane. </p> | ||
+ | <p> Conductivity with a biofilm is better with gold membranes, although the conductivity of gold membranes themselves isn't the best. This may be explained by the fact, that because of the thinner biofilm formation on gold membranes, the electrical wires touched not only the biofilm, but also the membrane, bypassing the biofilm and leading to imprecise measurements. </p> | ||
+ | <p> Approximate biofilm conductivity is therefore probably wrong for the gold membrane. However, it is interesting to notice that the biofilm conductivity measured for the bare alumina oxyde, PEDOT:Ts-coated one and PEDOT:PSS-coated give more or less the same value, suggesting that with more measurements, adapted equipement and better methods it would be indeed possible to measure the biofilm's conductivity with our PDMS well chips. </p> | ||
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Revision as of 00:09, 18 October 2018
RECONNECT NERVES
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Summary
Achievements:
- Successfully cloned a biobrick coding for secretion of NGF in pET43.1a and iGEM plasmid backbone pSB1C3, creating a new part BBa_K2616000.
- Successfully sequenced BBa_K2616000 in pSB1C3 and sent to iGEM registry.
- Successfully co-transformed E. coli with plasmid secreting proNGF and plasmid expressing the secretion system, creating bacteria capable of secreting NGF in the medium.
- Successfully characterized production of proNGF thanks to mass spectrometry and western blot.
- Successfully observed axon growth in microfluidic chip in presence of commercial NGF.
- Successfully observed activity of our proNGF in invitro cellular culture compared to commercial NGF with a concentration between 500 ng/mL and 900 ng/mL.
Next steps:
- Purify secreted proNGF, 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.
CELL CULTURE
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FIGHT INFECTIONS
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Summary
Achievements:
- Successfully cloned a biobrick 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. Although there was a high variability in our results, and we used several protocols to overcome it, in one case, we were able to observe a reduction in biofilm formation in the presence of our RIP.
Next steps:
- Clone the sensor device with inducible RIP production upon S. aureus detection.
- Improve the characterization of RIP effect on biofilm formation with a more standardized assay.
KILL SWITCH
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Summary
Achievements:
- Successfully cloned the biobrick Bba_K2616002 coding for toxin/antitoxin (CcdB/CcdA) system in pSB1C3, creating a new part.
- Successfully sequenced BBa_K2616002 in pSB1C3 and sent it to iGEM registry.
- Successfully observed normal growth 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.