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<p>Once checked, we cloned our construct into the <i>Escherichia coli</i> <b>BL21(DE3) pLysS</b> strain, a specific dedicated strain to produce high amounts of desired proteins under a T7 promoter. Bacteria were grown in 25 mL culture, and <b>protein expression</b> was induced with different IPTG concentrations during exponential phase at an OD<sub>600nm</sub> at 37°C. A 1 mL aliquot was centrifuged and the pellet stored at -20°C.<br> | <p>Once checked, we cloned our construct into the <i>Escherichia coli</i> <b>BL21(DE3) pLysS</b> strain, a specific dedicated strain to produce high amounts of desired proteins under a T7 promoter. Bacteria were grown in 25 mL culture, and <b>protein expression</b> was induced with different IPTG concentrations during exponential phase at an OD<sub>600nm</sub> at 37°C. A 1 mL aliquot was centrifuged and the pellet stored at -20°C.<br> | ||
− | After two hours induction, we centrifuged and collected both supernatant and pellet separately.<br><br></p></div> | + | After two hours of induction, we centrifuged and collected both supernatant and pellet separately.<br><br></p></div> |
Revision as of 17:19, 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.
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.
Next steps:
- Clone the sensor device with inducible RIP production upon S. aureus detection.
- Improve the characterization of RIP effect on biofilm formation.
KILL SWITCH
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Summary
Achievements:
- Successfully cloned a biobrick coding for toxin/antitoxin (CcdB/CcdA) system in iGEM plasmid backbone, creating a new part.
- Successfully observed survival 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.