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<div class="block two-third"> | <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 micrometers) and Sterlitech Alumina Oxide Membrane Filters (pores diameter of 0.2 micrometers).<br> | + | <p>The <b> membrane filter </b> is a key element of our prosthesis system, allowing the <b> confinement </b> of the genetically modified bacteria and the <b> conduction of neuron impulses </b>. We tested two types of membranes: Sterlitech Polycarbonate <b> Gold-Coated </b> Membrane Filters (pores diameter of 0.4 micrometers) and Sterlitech <b> Alumina Oxide </b> Membrane Filters (pores diameter of 0.2 micrometers).<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> | + | Sterlitech Alumina Oxide Membrane Filters were <b> coated </b> with different types of <b> biocompatible conductive polymers: PEDOT:PSS </b> (poly(3,4-ethylenedioxythiophene) polystyrene sulfonate), <b> PEDOT:Cl </b> and <b> PEDOT:Ts </b> .<br> |
− | To characterize the potential of the different types of membranes to be integrated into our prosthesis system, we | + | To characterize the potential of the different types of membranes to be integrated into our prosthesis system, <b> we designed a PDMS well chip for that exact purpose </b>, a modified <b>culture well </b>. The bottom of the well is a membrane, and a platinum wire touching the membrane electrically connects the inside of the well with the exterior.<br></p> |
</div> | </div> | ||
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<div class="legend"><b>Figure 23: </b> PEDOT:PSS </div> | <div class="legend"><b>Figure 23: </b> PEDOT:PSS </div> | ||
</div> | </div> | ||
+ | |||
+ | |||
+ | <div class="block separator-mark"></div> | ||
+ | |||
<div class="block full"> | <div class="block full"> | ||
− | <h2 style="text-align: left;"> | + | <h2 style="text-align: left;">Confinement</h2> |
− | <p></p> | + | <p> First requirement the membrane needs to meet is the ability to retain bacteria of the size of <i>E. coli</i> (1-2 micrometers). Theoretically, confinement should be garanteed because the membrane's pore size is smaller than <i>E. coli</i>. Three experiments were conducted on membrane microchannel chips to prove that the gold-coated membranes can retain bacteria. </p> |
+ | <h3> First experiment </h3> | ||
+ | <p> The optical density of an <i>E. coli</i> liquid culture was measured. Liquid culture was poured in a microchannel chip on one side, and optical density of the liquid that flowed to the other side of the microchannnels was measured. </p> | ||
+ | <h4 style="text-align: left;"> Results </h4> | ||
+ | <p> OD of liquid culture: 0.44 </p> | ||
+ | <p> OD of liquid after flowing through the chip: 0.41. </p> | ||
+ | <h4 style="text-align: left;"> Interpretation </h4> | ||
+ | <p> We expected a much lower OD after liquid flow through the chip, so this suggests the presence of a leak in the chip, that allows the liquid culture to flow without retaining the bacteria.</p> | ||
+ | </div> | ||
+ | |||
+ | <div class="block one-third"> | ||
+ | <h3> Second experiment </h3> | ||
+ | <p> A few drops of RFP expressing DH5alpha E. coli liquid culture were poured in a membrane microchannel chip. The chip was then observed under a microscope. </p> | ||
+ | <h4 style="text-align: left;"> Interpretation </h4> | ||
+ | <p> Membrane is located on the right side, and liquid culture was poured on that side, before the membrane. Bacteria was still able to flow to the left side, but they were not following the microchannels, instead they were just flowing in a single direction, suggesting the membrane lifts the microfluidic chip from below and thus causes massive leakings in the microfluidic circuitry. </p> | ||
</div> | </div> | ||
− | + | <div class="block two-third"> | |
+ | <img src="https://static.igem.org/mediawiki/2018/7/7b/T--Pasteur_Paris--Test-Filtre-RFP.jpg" style="width:500px"> | ||
+ | <div class="legend"><b>Figure 24: </b> Membrane microchannel chip under microscope</div> | ||
+ | </div> | ||
+ | |||
+ | <div class="block full"> | ||
+ | <h3> Third experiment </h3> | ||
+ | <p> A few drops of E. coli liquid culture were poured in a membrane microchannel chip. The chip was then observed under a microscope. </p> | ||
+ | <h4 style="text-align: left;"> Interpretation </h4> | ||
+ | <p> The membrane is located on the left side, and liquid culture was poured on that side, before the membrane. Bacteria this time wasn't able to flow to the right side, the membrane stopped their progression. It is clear on figure 25, that the left side is crowded with bacteria, and the right side is empty (apart from a few PDMS impurities). Final conclusion on the membrane microchannel chips is, that although the integration method of the membrane filter in the chip is complicated and a bit improvised, some chips apparently do fulfill their purpose, demonstrating this way the confinement of the bacteria with a membrane. Leaks observed in previous experiments were also probably caused by membrane filters that were not correctly stretching across the whole chip.</p> | ||
+ | </div> | ||
+ | |||
+ | <div class="block full"> | ||
+ | <img src="https://static.igem.org/mediawiki/2018/f/fd/T--Pasteur_Paris--Test-Filtre-2.jpg" style="width:700px"> | ||
+ | <div class="legend"><b>Figure 25: </b> Membrane microchannel chip under microscope with retaining membrane</div> | ||
+ | </div> | ||
+ | |||
+ | |||
+ | <div class="block separator-mark"></div> | ||
+ | |||
+ | |||
+ | |||
+ | <div class="block full"> | ||
+ | <h2 style="text-align: left;"> Polymer coating </h2> | ||
+ | <p> Bare alumina oxide membranes were coated with different polymers to enhance their conductivity values and their biocompatibility. </p> | ||
+ | </div> | ||
+ | |||
+ | |||
+ | <div class="block half"> | ||
+ | <img src="https://static.igem.org/mediawiki/2018/6/66/T--Pasteur_Paris--Alumina-oxide-membranes.jpg" style="width:400px"> | ||
+ | <div class="legend"><b>Figure 25: </b> White alumina oxide membranes before coating</div> | ||
+ | </div> | ||
+ | |||
+ | |||
+ | <div class="block half"> | ||
+ | <img src="https://static.igem.org/mediawiki/2018/f/f7/T--Pasteur_Paris--Alumina-oxide-membrane-micro.jpg" style="width:400px"> | ||
+ | <div class="legend"><b>Figure 25: </b> Scanning electron microscopy of bare alumina oxide membranes</div> | ||
+ | </div> | ||
+ | |||
+ | <div class="block full"> | ||
+ | <h3> PEDOT:PSS coating</h3> | ||
+ | <p> The color of the alumina oxide membranes changed radically from light grey to black, suggesting the deposit of PEDOT:PSS on the membrane, as expected. Scanning electron microscopy of a PEDOT:PSS-coated membrane revealed cluster-like formation of PEDOT:PSS deposits. It is thought that the lack of uniformity of the coating won't give the expected results in matters of biocompatibility and conductivity. </p> | ||
+ | </div> | ||
+ | |||
+ | <div class="block half"> | ||
+ | <img src="https://static.igem.org/mediawiki/2018/8/83/T--Pasteur_Paris--Coated-membranes-1.jpg" style="width:400px"> | ||
+ | <div class="legend"><b>Figure 25: </b> PEDOT:PSS-coated membranes</div> | ||
+ | </div> | ||
+ | |||
+ | <div class="block half"> | ||
+ | <img src="https://static.igem.org/mediawiki/2018/b/b5/T--Pasteur_Paris--PEDOT-PSS-membrane-micro.jpg" style="width:400px"> | ||
+ | <div class="legend"><b>Figure 25: </b> Scanning electron microscopy of PEDOT:PSS-coated membrane</div> | ||
+ | </div> | ||
+ | |||
+ | <div class="block full"> | ||
+ | <h3> PEDOT:Cl and PEDOT:Ts coating</h3> | ||
+ | <p> The color of the alumina oxide membranes changed radically from light grey to black with green shades for PEDOT:Cl and blue shades for PEDOT:Ts, suggesting the deposit of the polymers on the membranes, as expected. Scanning electron microscopy reveals a uniform thickening of the membrane's surface, suggesting a uniform PEDOT:Cl coating of the membrane. We expect better results from the PEDOT:Cl-coated and PEDOT:Ts-coated membranes than PEDOT:PSS-coated ones. </p> | ||
+ | </div> | ||
+ | |||
+ | <div class="block half"> | ||
+ | <img src="https://static.igem.org/mediawiki/2018/f/f2/T--Pasteur_Paris--Coated-membranes-3.jpg" style="width:400px"> | ||
+ | <div class="legend"><b>Figure 25: </b> PEDOT:Cl-coated membranes</div> | ||
+ | </div> | ||
+ | |||
+ | <div class="block half"> | ||
+ | <img src="https://static.igem.org/mediawiki/2018/3/33/T--Pasteur_Paris--Coated-membranes-2.jpg" style="width:400px"> | ||
+ | <div class="legend"><b>Figure 25: </b> PEDOT:Ts-coated membranes</div> | ||
+ | </div> | ||
+ | |||
+ | <div class="block half"> | ||
+ | <img src="https://static.igem.org/mediawiki/2018/0/05/T--Pasteur_Paris--PEDOT-membrane-micro.jpg" style="width:400px"> | ||
+ | <div class="legend"><b>Figure 25: </b> Scanning electron microscopy of PEDOT:Cl-coated membrane</div> | ||
+ | </div> | ||
+ | |||
+ | |||
+ | |||
+ | |||
+ | |||
+ | <div class="block separator-mark"></div> | ||
+ | |||
+ | |||
+ | |||
+ | <div class="block full"> | ||
<h2 style="text-align: left;">Conductivity</h2> | <h2 style="text-align: left;">Conductivity</h2> | ||
− | <p></p> | + | <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, as well as estimate the conductivity of a biofilm.</p> |
</div> | </div> | ||
− | + | <div class="block one-third"> | |
<img src="https://static.igem.org/mediawiki/2018/8/88/T--Pasteur_Paris--Well-chip.jpg" > | <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 class="legend"><b>Figure 24: </b> Hand-made PDMS well chip </div> | ||
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<div class ="block full"> | <div class ="block full"> | ||
− | <h3 style="text-align: left;"> | + | <h3>Platinum wire</h3> |
− | <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> | ||
+ | <img src="https://static.igem.org/mediawiki/2018/c/c4/T--Pasteur_Paris--Conductivity-platinum-wire.jpg" > | ||
+ | <div class="legend"><b>Figure 24: </b> Conductivity of a platinum wire for different frequencies </div> | ||
+ | <h4 style="text-align: left;"> Interpretation </h4> | ||
+ | <p> Voltage difference calculated is extremely low, indicating a very good conductivity for the platinum wires, so its resistance (in low frequencies) can be neglected at first glance when it will be used in PDMS well chips. Resistance increases in higher frequencies, because of the skin-effect in metals: the strip transforms into an antenna. But as we are going to use only low frequencies, this doesn't affect us. </p> | ||
+ | |||
</div> | </div> | ||
+ | |||
+ | |||
+ | <div class ="block full"> | ||
+ | <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 condu </p> | ||
+ | <h4 style="text-align: left;"> Results </h4> | ||
+ | <img src="https://static.igem.org/mediawiki/2018/c/c4/T--Pasteur_Paris--Conductivity-platinum-wire.jpg" > | ||
+ | <div class="legend"><b>Figure 24: </b> Conductivity of a platinum wire for different frequencies </div> | ||
+ | <h4 style="text-align: left;"> Interpretation </h4> | ||
+ | <p> Voltage difference calculated is extremely low, indicating a very good conductivity for the platinum wires, so its resistance (in low frequencies) can be neglected at first glance when it will be used in PDMS well chips. Resistance increases in higher frequencies, because of the skin-effect in metals: the strip transforms into an antenna. But as we are going to use only low frequencies, this doesn't affect us. </p> | ||
<div class="block two-third center"> | <div class="block two-third center"> |
Revision as of 23:10, 17 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.
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.