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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>Images from the imagery platform were analyzed on ImageJ. </p> | ||
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+ | <div class="legend"><b>Figure 13: </b> (Right) Percentage area of <FONT face="raleway">β</FONT>-III Tubulin in each well and (left) number of stained nuclei in each well with no NGF, 50 ng/mL, 250 ng/mL, 500 ng/mL, 750 ng/mL and 900 ng/mL of commercial NGF added in our medium Neurobasal, B27, GlutaMAX. Each condition was compared to the control group without NGF. (ns: non-significant, *: p<0.05, **: p<0.01, ***: p<0.001, ****: p<0.0001). </div> | ||
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+ | <p>As we can see in Figure 13 (right), 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. 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<exp>NTR</exp> is getting internalized, resulting in a decreasing number of available receptors. </p> | ||
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+ | <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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+ | <div class="legend"><b>Figure 14:</b> Ratio of the percentage area of <FONT face="raleway">β</FONT>-III Tubulin on the number of stained nucleus. <i>(ns: non-significant, * : p<0.05, ** : p<0.01, *** : p<0.001, **** : p<0.0001).</i> | ||
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Revision as of 23:12, 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.