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<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> | <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> | ||
<img src="https://static.igem.org/mediawiki/2018/b/bb/T--Pasteur_Paris--Electrical_circuit_1.pdf" style="width:500px"> | <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 | + | <div class="legend"><b>Figure 35: </b> Electric circuit used for the different conductivity measurements </div> |
+ | <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. </p> | ||
<h4 style="text-align: left;"> Results </h4> | <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"> | <img src="https://static.igem.org/mediawiki/2018/5/50/T--Pasteur_Paris--Membrane-Conductivity.jpg" style="width:500px"> | ||
− | <div class="legend"><b>Figure | + | <div class="legend"><b>Figure 36: </b> Conductivity of a platinum wire for different frequencies (mean value and standard deviation for each membrane) </div> |
<h4 style="text-align: left;"> Interpretation </h4> | <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> | <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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<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> | <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"> | <img src="https://static.igem.org/mediawiki/2018/8/84/T--Pasteur_Paris--Biofilm-Growth.jpg" style="width:500px"> | ||
− | <div class="legend"><b>Figure | + | <div class="legend"><b>Figure 37: </b> Biofilm growth (mean value and standard deviation for each type of membrane)</div> |
<h4 style="text-align: left;"> Results: biofilm conductivity </h4> | <h4 style="text-align: left;"> Results: biofilm conductivity </h4> | ||
− | <p> For conductivity measurements, we used the same electric circuit as in figure | + | <p> For conductivity measurements, we used the same electric circuit as in figure 35 . 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"> | <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 | + | <div class="legend"><b>Figure 38: </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> | <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"> | <img src="https://static.igem.org/mediawiki/2018/6/61/T--Pasteur_Paris--Biofilm-Conductivity.jpg" style="width:500px"> | ||
− | <div class="legend"><b>Figure | + | <div class="legend"><b>Figure 39: </b>Estimated biofilm conductivity </div> |
<h4 style="text-align: left;"> Interpretation </h4> | <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> 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> |
Revision as of 00:26, 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.