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<p>We gene synthesized the genetic construct of our kill-switch commercially. Once we received the sequence, called Seq9, in a commercial plasmid, we transformed competent bacteria <i>E. coli</i> DH5<FONT face="Raleway">α</FONT>. After bacterial culture and plasmid DNA extraction, we digested our DNA with restriction enzymes, extracted the inserts from the gel, and ligated it into linearized pSB1C3 for iGEM submission and expression in BL21(DE3).</p> | <p>We gene synthesized the genetic construct of our kill-switch commercially. Once we received the sequence, called Seq9, in a commercial plasmid, we transformed competent bacteria <i>E. coli</i> DH5<FONT face="Raleway">α</FONT>. After bacterial culture and plasmid DNA extraction, we digested our DNA with restriction enzymes, extracted the inserts from the gel, and ligated it into linearized pSB1C3 for iGEM submission and expression in BL21(DE3).</p> | ||
<p>We proved that our vector contained the insert by DNA electrophoresis (Figure 19).</p> | <p>We proved that our vector contained the insert by DNA electrophoresis (Figure 19).</p> |
Revision as of 22:19, 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.
- 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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BBa_K2616002
Sequencing results, when aligned to our original construct using Geneious, confirmed that pSB1C3 contained Seq9. This sequence was sent to the registry as Bba_K2616002.
The construction was successfully assembled. In Figure 20, we show that we used two different primers, allowing us to cover the whole sequence without mistakes. As visible, the mismatches are only present at the extremities of each primer sequencing. The final basepair identity is 100%.
Test of kill-switch efficiency
To test the efficiency of our kill-switch, we decided to cultivate transformed BL21(DE3) pLysS E. coli at several temperatures (15°C, 20°C, 25°C and 37°C). We used BL21(DE3) pLysS E. coli transformed with the empty pSB1C3 plasmid as the negative control. The bacteria growth was followed by measuring the optical density at 600 nm every 30 minutes for 6 hours, followed by two additional points at 18 hours and at 72 hours. Each experiment was done in triplicate and the standard deviation was calculated for every point. We showed that bacteria transformed with the kill-switch presented no measurable growth at 15°C and at 20°C during the 72 hours of the experiment, whereas the control population grew normally (Figure 21).
At 25°C, the kill-switch population grew more slowly than the control for the first 18 hours, but the growth eventually started to reach normal values at 72 hours.
Finally, at 37°C there was no difference in the growth of the kill-switch population compared to the control bacteria.
Thus, we successfully guarantee that our engineered bacteria will not be able to grow if they happened to be released in the environment.
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.