Team:Pasteur Paris/Demonstrate

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RECONNECT NERVES

Achievements:

  • Successfully cloned a part coding for secretion of NGF in pET43.1a and iGEM plasmid backbone pSB1C3, creating a new composite part BBa_K2616000
  • Successfully sequenced BBa_K2616000 in pSB1C3 and sent to iGEM registry
  • 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 characterized 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

DNA assembly



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 last 60 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 3).

Figure 1: proNGF and TEV production cassette

This DNA construct was ordered in two parts, named Seq1 (1096 bp) and Seq2 (1153 bp) in commercial plasmids pEX-A258 from gene synthesis. Seq1 and Seq2 were amplified in competent E. coli DH5-α. After bacteria culture and plasmid DNA extraction, we digested commercial vectors with restriction enzymes (NheI and BamHI for Seq1, MscI and HindIII for Seq2). We extracted the inserts from the gel and performed a ligation by using specific overlaps 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 pet43.1a contained Seq1 and Seq2 (Figure 2) and that pSB1C3 contained Seq1 and Seq2 (Figure 3) after digestion and DNA electrophoresis. Plasmid DNA of pSB1C3 construction was purified and sent for sequencing (Figure 4).

Figure 2: Agarose 1% gel after electrophoresis of digested pET43.1 containing Seq1 and Seq2 (Bba_K2616000) with NdeI. Colonies 6, 9, 10 ,11, 15 have the correct construction.
Figure 3: Agarose 1% gel after electrophoresis of digested pSB1C3 containing Seq1 and Seq2 (Bba_K2616000) with EcoRI/PstI. Colonies 3, 7 and 8 have the correct construction.

Alignment of Sequencing Results then confirmed that pSB1C3 contained Seq1 and Seq2.

Figure 4: Alignment of sequencing results for BBa_K2616000. Sequencing perform in pSB1C3 and three primers were designed (FOR1, FOR2, FOR3) to cover the whole sequence. Image from Geneious.

The construction was successfully assembled. On Figure 4, mismatches are visible which correspond to the reduced precision of sequencing after 600 bp. To avoid this lack of precision, we used three 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.

proNGF characterization and purification



Our chassis is Escherichia coliBL21(DE3) pLysS, a specific strain dedicated to producing high amounts of desired proteins under a T7 promoter. Thus, we co-transformed our bacteria with BBa_K2616000 and pVDL 9.3, generously provided by Dr. Victor de Lorenzo, from Centro Nacional de Biotecnologia of Madrid, bearing HlyB and HlyD (Type I secretion system) sequences, in order to get a chance to secrete NGF out of the cell.

Bacteria were grown at a large scale (800 mL), and proNGF expression was induced with 0.1 mM IPTG for 2 hours at 37°C.

We tried to achieve His-tagged proNGF purification using Ni-NTA affinity purification column. We eluted our protein using a gradient of imidazole-containing buffer and one peak was detected.

Figure 5: FPLC proNGF purification with ÄKTA pure (General Electric) Ni-NTA column was equilibrated with buffer A (50 mM Tris, pH 7.4, 200 mM NaCl). Supernatant of lyzed bacteria was introduced through the column. Washing with 5% of buffer B. Elution by buffer B gradient (buffer A + imidazole 250 mM). UV absorbance at 280nm is shown in blue, conductivity in red, and concentration of buffer B in green.

We analyzed bacterial lysate and purification fractions using SDS-PAGE electrophoresis and Mass spectrometry.

Figure 6: SDS-PAGE gel Bis-Tris 4-12% of bacterial lysate and proNGF purification fraction by SDS-PAGE.

The proNGF purification using NiNTA column is not conclusive. Many proteins are found on elution fractions. His-tagged proNGF fused to HlyA export signal should be found at 33 kDa while the proNGF cleaved by TEV protease should be found at 27 kDa. We finally analyzed five gel bands of the FPLC flow-through (lane 2, Figure 6) by mass spectrometry, by LC/MS/MS to verify the presence of proNGF.

According to Figure 7, proNGF pattern are found on each lane sent to mass spectrometry. The major amount is found on fraction 5, corresponding to 33 kDa, at this molecular weight, the proNGF is still fused to the signal export. The TEV protease, 34 kDa fused to signal export and 28 kDa cleaved from the signal export are found.

Figure 7: Distribution of proNGF and TEV protease by gel fractions after mass spectrometry analysis.

Analysis of Fraction 5 of the gel shows our protein proNGF is present but is still bound to its signal peptide HlyA. (Figure 8) Mass spectrometry spectrum of Peptide A, IDTACVCVLSR, from proNGF sequence is shown in Figure 9. Mass spectrometry spectrum of Peptide B, IISAAGSFDVKEER from fused HlyA signal export is shown in Figure 9. The presence of mass spectrometry identified peptides corresponding to the fusion of proNGF and HlyA indicate some proNGF uncleaved from the signal export

Figure 8: Alignment sequences of proNGF fused to HlyA export signal and peptides identified by mass spectrometry. Sequence have been annotated to match corresponding protein.
Figure 9: Mass spectrometry spectrum. A) Peptide identified corresponding to proNGF. B) Peptide identified corresponding to the fusion of proNGF and HlyA export signal.

The proNGF did not seem to be retained on the affinity column. We performed batch purification using NiNTA beads under native and partial denaturing conditions (Urea 2 M) followed by Western Blot analysis with immunodetection through Anti-His Antibodies Alexa Fluor 647. (Figure 10) Detection of His-tag in the pellet supernatant of induced BL21 with 1 mM IPTG and flow through when partially denatured.

His-tagged proNGF was not retained on NiNTA beads. N-terminal His tag may be hidden in the protein fold. Consequently, we did not manage to purify the proNGF.

Figure 10: Western Blot analysis of batch purification proNGF under native and partial denaturing conditions.

FIGHT INFECTIONS


Achievements:

  • Successfully cloned a part coding for RIP in pBR322 and in pSB1C3, creating a new part.
  • Successfully cultivated S. aureus biofilms 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.

The sequence we designed contains two RIP (RNAIII Inhibiting Peptide) sequences fused to two different export signal peptides 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 by gene synthesis, in order to have more DNA, we transformed competent bacteria E. coli DH5-alpha resulting in clones. 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.

Figure 1: 6% Agarose gel after electrophoresis of digested pSB1C3 containing Seq8 (Bba_K2616001). All colonies except 1, 3 and 7 have the insert.

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 1).
Sequencing confirmed that it was the correct sequence.

Once checked, we cloned our construct into the Escherichia coli BL21(DE3) pLysS 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 have enteres in a phase of exponential growth (approximately at 0.8 OD 600 nm) at 37°C.
After two hours induction, we centrifuged and collect supernatant and pellet separately.

Fluorescence reading experiments



Since RIP is only a seven-aminoacid peptide, we were not able to check its production by classic SDS-PAGE. Thus, we tried to check its expression by observing its effect on Staphylococcus aureus growth and adhesion. We grew a S. aureus strain expressing GFP (Green Fluorescent Protein), gently provided by Dr. Jean-Marc Ghigo on 96-well microtiter plates with different fractions of supernatant or pellet of our BL21(DE3) pLysS 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 performed such an experiment several times, and the results were not always concluding, very likely because of bias in the fluorescence reading on the plate or because of a too damaged biofilm after the washing step.

Crystal violet staining



Since, fluorescence measurements were not concluding, we tried to improve our quantification biofilm formation by Crystal violet coloration and then measuring absorbance at 570 nm. Again, the results were very heterogeneous between our different experiments.

Figure 2: Biofilm assay with crystal violet staining

Biofilm PFA fixation before staining



With more time, we would certainly have been able to optimize our protocol to best fit with the strain we use, but for the time being, there are still few concluding results.

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 7: Agar gel after electrophoresis of digested pSB1C3 containing Seq9 (Bba_K2616002) in columns 6 to 11. Colonies 2 and 6 have the correct plasmid.

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 8: Effect of different temperatures on the growth of Cryodeath kill-switch transformed BL21 E. coli

Thus, we successfully guarantee that our engineered bacteria will not be able to grow if they happened to be released in the environment.