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<p><i>Imaging was performed in collaboration with the BioImagerie Photonique platform of the Institut Pasteur. Data are presented as MEAN ± SEM. Significance between 2 different groups was determined using an Ordinary one-way ANOVA test on the software Prism6 (GraphPad). (ns: non-significant, *: p<0.05, **: p<0.01, ***: p<0.001, ****: p<0.0001) </i> </p> | <p><i>Imaging was performed in collaboration with the BioImagerie Photonique platform of the Institut Pasteur. Data are presented as MEAN ± SEM. Significance between 2 different groups was determined using an Ordinary one-way ANOVA test on the software Prism6 (GraphPad). (ns: non-significant, *: p<0.05, **: p<0.01, ***: p<0.001, ****: p<0.0001) </i> </p> | ||
<p>As an alternative to our recombinant proNGF for control experiments, we performed an <i>in vitro</i> neural primary culture with commercial NGF. For this, a pair of E18 Sprague Dawley cortexes were purchased from BrainBits.co.uk. We digested the tissue with manufacturer provided papain according to their protocol and seeded 40 000 dissociated neurons on our microfluidic chips with different conditions of culture for six days at 37°C, and 5% CO2. </p> | <p>As an alternative to our recombinant proNGF for control experiments, we performed an <i>in vitro</i> neural primary culture with commercial NGF. For this, a pair of E18 Sprague Dawley cortexes were purchased from BrainBits.co.uk. We digested the tissue with manufacturer provided papain according to their protocol and seeded 40 000 dissociated neurons on our microfluidic chips with different conditions of culture for six days at 37°C, and 5% CO2. </p> | ||
+ | |||
<p>On our two-chamber microfluidic devices, we seeded neurons only on one side. Fifteen chips were used in total. After six days, neurons are fixed with paraformaldehyde (PFA) 4% and stained with DAPI. For differentiated markers: MAP2 (coupled with Alexa Fluor 555), a cytoskeletal associated protein and Beta-III Tubulin (coupled with Alexa Fluor 488), one of the major components of microtubules and a neuron-specific marker were used.</p> | <p>On our two-chamber microfluidic devices, we seeded neurons only on one side. Fifteen chips were used in total. After six days, neurons are fixed with paraformaldehyde (PFA) 4% and stained with DAPI. For differentiated markers: MAP2 (coupled with Alexa Fluor 555), a cytoskeletal associated protein and Beta-III Tubulin (coupled with Alexa Fluor 488), one of the major components of microtubules and a neuron-specific marker were used.</p> | ||
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<img src="https://static.igem.org/mediawiki/2018/4/43/T--Pasteur_Paris--Figure_11.png"> | <img src="https://static.igem.org/mediawiki/2018/4/43/T--Pasteur_Paris--Figure_11.png"> | ||
− | <div class="legend"><b>Figure 11: </b> In orange, are displayed bacteria found inside one of our microfluidic | + | <div class="legend"><b>Figure 11: </b> In orange, are displayed bacteria found inside one of our microfluidic devices.</div> |
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− | <p>Because we were running out of fresh microfluidic chips, and since we already proved that our device was working as expected, we switched and put our next cultures in a 96-well plate for 10 days at 37°C, testing the influence of the different concentrations of NGF on the growth of the cells. </p> | + | <p>Because we were running out of fresh microfluidic chips, and since we already proved that our device was working as expected, we switched and put our next cultures in a 96-well plate for 10 days at 37°C, testing the influence of the different concentrations of NGF on the growth of the cells (Figure 14). </p> |
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− | <p>As we can see in Figure | + | <p>As we can see in Figure 15 <b>(A)</b>, it was possible to observe a difference in the percentage of area taken by the <FONT face="raleway">β</FONT>-III Tubulin. Indeed, it is possible to observe a significant increase in this percentage when commercial NGF is put at a concentration of 250 ng/mL or higher. The concentration of NGF seems to influence the growth of axons. It was possible to observe the same significant increase of cell number at a concentration of 250 ng/mL or higher (Figure 15 <b>B</b>). We can also see that at a concentration of 900 ng/mL, it seems that both the percentage of area taken by the <FONT face="raleway">β</FONT>-III Tubulin and the number of cells decrease, even though it is not significant, we can hypothesize that at a high concentration of NGF, the receptor p75<sup>NTR</sup> is getting internalized, resulting in a decreasing number of available receptors. </p> |
<p>It seems that commercial NGF has a dose-response effect on both the growth of neuronal axons and / or the survival of the cells. To determine in which category the NGF was affecting, we standardize the percentage area of <FONT face="raleway">β</FONT>-III Tubulin compared to the number of cells. </p> | <p>It seems that commercial NGF has a dose-response effect on both the growth of neuronal axons and / or the survival of the cells. To determine in which category the NGF was affecting, we standardize the percentage area of <FONT face="raleway">β</FONT>-III Tubulin compared to the number of cells. </p> | ||
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− | <p>As we can see in figure | + | <p>As we can see in figure 16, we have a decreasing amount of <FONT face="raleway">β</FONT>-III Tubulin per nuclei each time the concentration of NGF gets higher. We can see a significant decrease of this ratio when the NGF is at 500 ng/mL and higher, which is not an expected result and an opposite result from the images that we occurred from the platform. </p> |
− | <p>We assumed from the start that all of our cells put in culture were neuronal cells, which might not be the case. We know that the NGF has an effect of the survival of the cells <sup>[1], [2]</sup> (Figure | + | <p>We assumed from the start that all of our cells put in culture were neuronal cells, which might not be the case. We know that the NGF has an effect of the survival of the cells <sup>[1], [2]</sup> (Figure 15 <b>B</b>). We did not have the suitable marker to differentiate the neuronal cells from the other types of cells, and should have stained the cells with NeuN, a neuronal nuclear antigen used as a biomarker for neurons. Therefore, the standardization we did with the number of cells is not an accurate one. We can still appreciate the qualitative results we had (Figure 14 and 15 <b>A</b>) and are positive on the effect NGF has on axon’s growth as well as cell survival.</p> |
− | <p>After having collected the data on the effect of commercial NGF, we decided to put in culture our cells in the presence of our bacterial lysate to test the effect of our proNGF. We put in culture for 2 days 30 000 cells with or without commercial NGF at 500 ng/mL and 900 ng/mL as well as our bacterial lysate in different dilutions. Since we wanted to inactivate as much bacterial proteins as possible ( | + | <p>After having collected the data on the effect of commercial NGF, we decided to put in culture our cells in the presence of our bacterial lysate to test the effect of our proNGF. We put in culture for 2 days 30 000 cells with or without commercial NGF at 500 ng/mL and 900 ng/mL as well as our bacterial lysate in different dilutions. Since we wanted to inactivate as much bacterial proteins as possible (endotoxins), we checked the denaturation temperature for our proNGF, 70°C, and heat-inactivated the lysate at 60°C for 5 minutes before putting it in culture. Due to lack of time, only one well per condition was analyzed. </p> </div> |
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<img src="https://static.igem.org/mediawiki/2018/d/d1/T--Pasteur_Paris--Figure_17B.jpg"style="max-width:30em;"></div> | <img src="https://static.igem.org/mediawiki/2018/d/d1/T--Pasteur_Paris--Figure_17B.jpg"style="max-width:30em;"></div> | ||
− | <div class="legend"><b> Figure 17:</b> (A) Percentage area of <FONT face="raleway">β</FONT>-III Tubulin in each well and (B) percentage area of nucleus in each well with no commercial NGF, 500 ng/mL or 900 ng/mL or bacterial lysate at 1/5, 1/10, 1/20 or 1/30 added in our medium Neurobasal, B27, GlutaMAX. </div> | + | <div class="legend"><b> Figure 17:</b> <b>(A)</b> Percentage area of <FONT face="raleway">β</FONT>-III Tubulin in each well and <b>(B)</b> percentage area of nucleus in each well with no commercial NGF, 500 ng/mL or 900 ng/mL or bacterial lysate at 1/5, 1/10, 1/20 or 1/30 added in our medium Neurobasal, B27, GlutaMAX. </div> |
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− | <p>We can see in Figure | + | <p>We can see in Figure 17 that our lysate seems to increase the percentage area of the <FONT face="raleway">β</FONT>-III Tubulin compared to the control without NGF. Our results with the commercial NGF seem to be equivalent to the results we had from our first experiment (Figure 15), with a decrease of axons at a concentration of 900 ng/mL. We can hypothesize that the lysate does have an effect on axon’s growth from the increasing percentage area of <FONT face="raleway">β</FONT>-III Tubulin, increase similar to the one we observe in our first experiment (Figure 15) and that the activity of our proNGF could be equivalent to commercial NGF with a concentration between 500 ng/mL and 900 ng/mL.</p> |
− | <p>We also could see an influence of the commercial NGF on the survival of the cells, similar to our first experiment (Figure | + | <p>We also could see an influence of the commercial NGF on the survival of the cells, similar to our first experiment (Figure 15). Our lysate, put at a concentration of 1/10 and higher, seems to have the same effect (Figure 18). </p> |
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Revision as of 02:54, 18 October 2018
RECONNECT NERVES: DNA ASSEMBLY
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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 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.
Next steps:
- Purify secreted proNGF, and characterize its effects on neuron growth thanks to our microfluidic device.
RECONNECT NERVES: CELL CULTURE
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Summary
Achievements:
- 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:
- Statistical analysis of our in vitro culture in presence of bacterial lysate.
- 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.
MEMBRANE
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Summary
Achievements:
- Successfully demonstrated the confinement of bacteria by a membrane filter.
- Successfully coated alumina oxide membranes with PEDOT:Cl and PEDOT:Ts .
- Partially coated alumina oxide membranes with PEDOT:PSS.
- Successfully demonstrated the enhanced conductivity induced by the PEDOT:Cl and PEDOT:Ts coating.
- Successfully enhanced biocompatibilty with PEDOT:Cl coating.
Next steps:
- Enhance measurement precision for membrane conductivity with and without biofilm.
- Improve PEDOT:PSS coating to form a uniform layer.