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<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>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 (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> | + | <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 (produced with <a href="http://parts.igem.org/Part:BBa_K2616000"> Bba_K2616000 </a> |
+ | 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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Revision as of 03:47, 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.