Difference between revisions of "Team:Pasteur Paris/Results"

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<p>Once we received the sequences encoding for this production cassette (named construction Seq9) in commercial plasmids, in order to have more DNA, we transformed competent bacteria <i>E. coli</i> DH5alpha resulting in clones. After bacteria culture and plasmid DNA extraction, we digested commercial vectors with restriction enzymes, extracted the inserts from the gel, and ligated it into linearized pSB1C3 for iGEM submission and expression in BL21(DE)3.</p>
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                <p>We repeated the procedure (transformation in <i>E. coli</i> Stellar competent cells, bacteria culture, plasmid DNA extraction, digestion) and we proved that our vector contained the insert by electrophoresis.</p>
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<div class="legend"><b>Figure 6: </b> </div>
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<p>To test the efficiency of our kill-switch, we decided to cultivate BL21(DE)3 E. coli transformed with it at several temperatures (15°C, 20°C, 25°C and 37°C). The growth was followed by measuring the optical density at 600nm every 30 minutes for 6 hours, followed by two additional points at 18 hours and at 72 hours. Each experiment was done in a triplicate and the standard deviations were calculated for every point.  We show that the bacteria transformed with the kill-switch showed <b>no measurable growth</b> at 15°C and at 20°C during the 72 hours of the experiment, whereas the control population grew normally.</p>
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                <p>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. </p>
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                <p>Finally, at 37°C there was no difference in the growth of the kill-switch population compared to the control bacteria. </p>
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<div class="legend"><b>Figure 7: </b> </div>
 
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Revision as of 16:19, 30 September 2018

""

FIGHT INFECTIONS


Achievements:

  • Successfully cloned a part coding for RIP in pBR322 and in pSB1C3, creating a new composite part
  • Successfully cultivate S. aureus biofilms with different supernatants

Next steps:

  • Clone the sensor device with inducible RIP production upon S. aureus detection
  • Prove …

The sequence we designed contains two RIP (RNAIII Inhibiting Peptide) sequences fused to two different export signal peptide for E. coli Type II Secretion System: DsbA and MalE, placed in N-terminal. (image: Figure 1. Schematic representation of the RIP production cassette. The cassette is composed of RIP sequence (blue) fused to DsbA signal (green) and further RIP sequence again (green) fused to MalE signal (red).)

Once we received the sequence encoding for this production cassette (named construction Seq8, size 461bp) in commercial plasmid, in order to have more DNA, we transformed competent bacteria E. coli DH5alpha resulting in clones (photo). After bacteria culture and plasmid DNA extraction, we digested commercial vector with EcoRI and PstI restriction enzymes, extracted the insert from the gel, and ligated it into linearized pSB1C3 for RIP expression.
We repeated the procedure (transformation in E. coli Stellar competent cells, bacteria culture, plasmid DNA extraction, digestion) and we proved that our vector contained the insert by electrophoresis (photo).
Sequencing confirmed that it was the correct sequence (photo).

Once checked, we cloned our construct into the Escherichia coli BL21(DE3) strain, a specific dedicated strain to produce high amounts of desired proteins under a T7 promoter. Bacteria were grown in 25 mL culture, and protein expression was induced with IPTG when bacteria are in a phase of exponential growth at 37°C.
After two hours induction, we centrifuged and collect supernatant and pellet separately.

Since RIP is only a seven-aminoacid peptide, we were not able to check its production by SDS-Page. Thus we tried to check its expression by observing its effect on Staphylococus aureus growth and adhesion. We grew a S. aureus strain expressing GFP (Green Fluorescent Protein) on 96-well microtiter plates with different fraction of supernatant or pellet of our BL21(DE3) bacterial cultures.

After 48h or more incubation, we washed the plates in order to discard planktonic bacteria, and read fluorescence (excitation at 485 nm and measuring emission at 510 nm).

We also quantified biofilm formation by Crystal violet coloration, and then measuring absorbance at 570 nm.

RECONNECT NERVES

Achievements:

  • Successfully cloned a part coding for secretion of NGF in pET43.1a and iGEM plasmid backbone, creating a new composite part
  • Successfully co-transform E. coli with plasmid secreting NGF and plasmid expressing the secretion system, creating bacteria capable of secreting NGF in the medium
  • Successfully characterize production of NGF thanks to mass spectrometry
  • Successfully observe axon growth in microfluidic chip in presence of commercial NGF

Next steps:

  • Purify secreted NGF, 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

The sequence we designed codes for two different proteins: proNGF (Nerve Growth Factor) and TEV protease (from Tobacco Etch Virus). These two proteins are fused in C-terminal with a signal peptide for E. coli Type I Secretion System which consists in the 60 last amino-acids of HaemolysinA (HlyA). Each coding sequence is separated from the signal peptide by the cleavage sequence for TEV, in order to get the protein without its signal peptide.

Figure 1: proNGF and TEV production cassette

Once we received the sequences encoding for this production cassette (named constructions Seq1, size 1096 bp and Seq2, size 1153bp) in commercial plasmids, in order to have more DNA, we transformed competent bacteria E. coli DH5alpha resulting in clones. After bacteria culture and plasmid DNA extraction, we digested commercial vectors with restriction enzymes (NheI and BamHI for Seq1, MscI and HindIII for Seq2), extracted the inserts from the gel, and ligated it into linearized pET43.1a for proNGF expression and into pSB1C3 for iGEM sample submission.
We repeated the procedure (transformation in E. coli Stellar competent cells, bacteria culture, plasmid DNA extraction, digestion) and we proved that our vector contained the insert by electrophoresis.

Figure 2: Agar gel after electrophoresis of digested pSB1C3 containing Seq1 and Seq2 (Bba_K…). Colonies 3,7 and 8 have the correct plasmid, colonies 4 and 10 may only have one of the two inserts.

Sequencing then confirmed that it was the correct sequence.

Once checked, we cloned our construct into the Escherichia coli BL21(DE3) strain, a specific dedicated strain to produce high amounts of desired proteins under a T7 promoter. Bacteria were grown on large scale (800 mL), and proNGF expression was induced with 0.1 mM IPTG for 2 hours at 37°C.

We tried to achieve protein purification using the His-Tag. We resort to a Ni-NTA purification column for purification to work. We eluted our protein using a gradient of imidazole-containing buffer and one peak was detected.

Figure 3: FPLC protein purification

We checked the presence of proteins in the fractions by SDS-PAGE. We clearly noted the appearance of bands at about …, while the expected size of our protein is 27 kDa if the signal peptide has been cleaved and 33 kDa if it is still bound. We hypothesized that it could be …

Figure 4: Protein elution profile of proNGF After lysis and FPLC, fractions ran on SDS-PAGE . SN : supernatant. FT : flowthrough. Lanes 1 to 6 : fractions. C2 monomers (25 kDa) and dimmers (50 kDa) shown by dark arrows. MW : molecular weight marker.
We also send an extract of resuspended pellet to be analyzed by mass spectrometry. We obtained the following results:
Figure 5:

KILL SWITCH

Achievements:

  • Successfully cloned a part coding for toxin/antitoxin (CcdB/CcdA) system in iGEM plasmid backbone, creating a new composite part
  • Successfully observe 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

Once we received the sequences encoding for this production cassette (named construction Seq9) in commercial plasmids, in order to have more DNA, we transformed competent bacteria E. coli DH5alpha resulting in clones. After bacteria culture and plasmid DNA extraction, we digested commercial vectors with restriction enzymes, extracted the inserts from the gel, and ligated it into linearized pSB1C3 for iGEM submission and expression in BL21(DE)3.

We repeated the procedure (transformation in E. coli Stellar competent cells, bacteria culture, plasmid DNA extraction, digestion) and we proved that our vector contained the insert by electrophoresis.

Figure 6:

To test the efficiency of our kill-switch, we decided to cultivate BL21(DE)3 E. coli transformed with it at several temperatures (15°C, 20°C, 25°C and 37°C). The growth was followed by measuring the optical density at 600nm every 30 minutes for 6 hours, followed by two additional points at 18 hours and at 72 hours. Each experiment was done in a triplicate and the standard deviations were calculated for every point. We show that the bacteria transformed with the kill-switch showed no measurable growth at 15°C and at 20°C during the 72 hours of the experiment, whereas the control population grew normally.

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.

Figure 7: