Team:Pasteur Paris/Results

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RECONNECT NERVES

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 Escherichia 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 1).

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, BBa_K2616000 .

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 E. coli BL21(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 fractions 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 fraction 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. In light blue peptides that match proNGF amino acids sequence. In light yellow, peptides that match HlyA signal export. Sequence has been annotated to match corresponding protein amino acid sequences : In orange His tagged proNGF, in red TEV protease cleaving site, in rose HlyA signal export.
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.

Achievements:

  • Successfully cloned a part 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-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.

FIGHT INFECTIONS


RIP Secretion BBa_K2616001



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 on 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).(Figure 11)

Figure 11: proNGF and TEV production cassette

Once we received the sequence encoding for this production cassette named Seq8 (461bp) in commercial plasmid pEX-A258 by gene synthesis. Plasmids was amplified in competent E. coli DH5alpha. After bacteria culture and plasmid DNA extraction, we digested commercial vector with EcoRI and PstI restriction enzymes. We extracted the inserts from the gel and performed a ligation by using specific overlaps into linearized pBR322 for RIP 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 vectors contained the insert by electrophoresis (Figure 12,13).

Figure 12: Agarose 1% gel after electrophoresis of digested pSB1C3 containing Seq8 (Bba_K2616001) with PstI and EcoRI. All colonies except 1, 3 and 7 contained the insert.
Figure 13: Agarose 1% gel after electrophoresis of digested pBR322 containing Seq8 (Bba_K2616001) with NdeI (lane 1 to 7) All colonies except colonies 2 and 7 contained the insert.

Alignment of Sequencing Results then confirmed that pSB1C3 contained Seq8, Bba_K2616001 .

Figure 14: Alignment of sequencing results for BBa_K2616001. Sequencing perform in pSB1C3 plasmid and one primer was designed (FOR1) to cover the whole sequence. Image from Geneious. Pairwise % Identity: 100%.

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 different IPTG concentration when bacteria have entered in a phase of exponential growth (approximately at 0.8 OD 600 nm) at 37°C. Pellet was sonicated and supernatant was kept
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 containing BBa_K26160001.

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).

Figure 15: Measurement of GFP fluorescence from S. aureus biofilms cultivated with different IPTG induction concentrations of RIP peptide. Every measure was done eight times and the bars show the average fluorescence. CM= Culture Medium from the induced E. coli culture.. SL = Lysis Supernatant from the induced E. coli culture.

Some of the results we got were extremely encouraging. For example, figure 15 shows an average 3-fold reduction of fluorescence from S. aureus biofilms when they were cultivated in presence of the bacterial lysate of an induced culture of BL-21 E. coli transformed with BBa_K2616001.

However, we performed experiments several times, and the results were not always as concluding. This variability is very likely due to a bias due regarding different approaches used for supernatant removal and washes. When using the flicking approach, we damaged our biofilm. Then, we removed planktonic cells by micropipette.

Crystal violet staining



Since fluorescence measurements were not satisfying enough, we tried to improve our methods to quantify biofilm formation. Thus, we began to color biofilms by Crystal violet 0.1% staining and measuring absorbance at 570 nm. Again, the results were very heterogeneous between our different experiments, and between the different protocols. For instance, we tried to compare our protocol to WPI Worcester team's staining protocol, and the results, given in Figure 16, significantly differ.

Figure 16: Measurement of absorbance at 570 nm S. aureus biofilms cultivated with different IPTG induction concentrations of RIP peptide and stained with Crystal violet. Every measure was done eight times and the bars show the average fluorescence. CM= Culture Medium from the induced E. coli culture.. SL = Lysis Supernatant from the induced E. coli culture.

Biofilm PFA fixation before staining



We wanted to avoid biofilm damage or loss during these steps. In order to do that, we used Bouin solution to fix the formed biofilm after 24 and 48 hours of culture. (Figure 17) Then biofilms were either stained with Crystal Violet 0.1% and resuspended in acetic acid 30% or resuspended in PBS 1X. Surprisingly, with this method biofilm formation was higher when cultivated with cell extracts containing RIP. For now, we are not able to explain why.

Figure 17: Biofilm culture fixed with Bouin's solution in 96-well micrometer plate

With more time, we would certainly have been able to optimize our protocols to best fit with the strain we use, but for the time being, we are not able to give a final conclusion on whether or not our RIP peptide inhibits S. aureus biofilm formation.

S. aureus Detection and RIP secretion BBa_K2616003



The sequence we designed contains the agr detection system from S. aureus and secretion of 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.(Figure 18)

Figure 18: S. aureus sensor device and RIP production cassette

Once we received the sequence encoding for this production cassette, named Seq5 (1422 bp), Seq6 (960 bp) and Seq7 (762 bp) in commercial plasmid pEX-A258 by gene synthesis. Plasmids were amplified in competent E. coli DH5alpha.

After bacterial culture and plasmid DNA extraction, we digested the commercial vector with XbaI and BamHI for Seq5, MscI, and SphI for Seq6, HindII, and SpeI for Seq7 restriction enzymes. We extracted the insert from the gel and ligated by specific overlaps into linearized pBR322 for expression and into pSB1C3 for iGEM sample submission.

We had trouble to proceed the ligation of the three inserts to linearized pBR322 and pSB1C3. We discussed with Takara Bio about our ligation issues, the GC percentage on our overlaps was too high to allow a good ligation. Due to the lack of time, we were not able to redesign the overlaps for this construction.

Achievements:

  • Successfully cloned a part 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

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 DNA electrophoresis.(Figure 19)

Figure 19: Agar gel after electrophoresis of digested pSB1C3 containing Seq9 (Bba_K2616002) in columns 6 to 11. Colonies 2 and 6 have the correct plasmid.

Alignment of Sequencing Results then confirmed that pSB1C3 contained Seq9, Bba_K2616002 .

Figure 20: Alignment of sequencing results for BBa_K2616002. Sequencing perform in pSB1C3 and two primers were designed (FOR1 and FOR2) to cover the whole sequence. Image from Geneious. Pairwise Identity: 96.9%.

The construction was successfully assembled. On Figure 20, mismatches are visible which correspond to the reduced precision of sequencing after 600 bp. To avoid this lack of precision, 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.

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. (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.

Figure 21: 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.

Achievements:

  • Successfully cloned a part 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.

Membrane

The membrane filter is a key element of our prosthesis system, allowing the confinement of the genetically modified bacteria and the conduction of neuron impulses. We tested two types of membranes: Sterlitech Polycarbonate Gold-Coated Membrane Filters (pores diameter of 0.4 micrometers) and Sterlitech Alumina Oxide Membrane Filters (pores diameter of 0.2 micrometers).
Sterlitech Alumina Oxide Membrane Filters were coated with different types of biocompatible conductive polymers: PEDOT:PSS (poly(3,4-ethylenedioxythiophene) polystyrene sulfonate), PEDOT:Cl and PEDOT:Ts.
To characterize the potential of the different types of membranes to be integrated into our prosthesis system, we evaluated the coating of the alumina oxide membranes, their biocompatibility and their electrical conductivity.

Figure 23: PEDOT:PSS

Biocompatibility

Conductivity

Figure 24: Hand-made PDMS well chip

The conductivity of the membranes was measured on a self-made device. It consists of a culture well made of PDMS (polydimethylsiloxane), with a membrane filter at its bottom and a platinum wire linking the conductive membrane filter with the exterior.

Platinum wire

The voltage difference between the two extremities of the wire was measured.

Figure 25: Voltage difference between the two extremities of the platinum wire.

The voltage difference between different platinum wires is pretty much the same. As we want to compare the differences between multiple membranes, we don't need to take into account the variability from one chip to another of the platinum wire's resistance. That means, it is meaningful to measure the voltage difference between a point on the membrane and the extremity of the wire outside the well and use this data to compare the membranes.

Membranes

The voltage difference between a point on the membrane (located near the border of the membrane filter) and the extremity of the platinum wire outside the well was measured.

Figure 26: Voltage difference between the extremity of the platinum wire outside the well and a point on the membrane.

CELL CULTURE

Neuron culture

Waiting for an alternative on our proNGF, we performed an in vitro neural primary culture with commercial NGF. For this, we bought from the company BrainBits a Sprague Dawley E18 cortex pair. We digested the tissue with papain according to their protocol and seeded 40 000 dissociated neurons on our microfluidic chips with different condition of culture for 6 days at 37°C 5% CO2.

Neurons were seeded only on one side of our device. After 6 days, neurons are fixed with Paraformaldehyde 4% and stained with DAPI and for differentiated markers: MAP2 (coupled with Alexa Fluor 555), a cytoskeletal associated protein and Beta-III Tubuline (coupled with Alexa Fluor 488), one of the major component of microtubules and a neuron-specific marker.

We can see in Figure 27 that we had contaminations of our microfluidic chips and most of our experiments could not be analyzed, except for a few microfluidic chips displayed in Figure 28.

Figure 27: Bacteria found in our microfluidic device during cell culture (bacteria are in orange)
Figure 28: Sprague Dawley E18 cortex neurons after 6 days of incubation at 37°C, 5% CO2. Blue: Nucleus, Green: Beta-III Tubuline, Yellow: Co-localization of Beta-III Tubuline and MAP2. (A) Neurons were put in culture in Neurobasal, B27, GlutaMAX. (B) Neurons were put in culture in DMEM FBS 10%.

As we can see, we succeeded in growing the cells inside our device in presence of Neurobasal, B27, and GlutaMAX. It is possible to see neurons passing through one chamber to the other in this experiment. Unfortunately, PDMS of the microfluidic chips detached from the bottom of the glass culture dish, leading to the growth of cells, not inside the microchannel but below them.

Growth in presence of commercial NGF

Neurons were put in culture in presence of commercial NGF at different concentration: 50 ng/mL, 250 ng/mL, 500 ng/mL, 750 ng/mL and 900 ng/mL. Optimal concentration was determined thanks to the modeling of NGF diffusion inside the medium. It was possible to capture the cells passing through one chamber of the microfluidic chip to other during a time lapsed using phase-contrast realized for the first 48h of culture at the Photometric BioImagery platform, proving that our device was working as expected.

Figure 29: Neuron entering the microchannel are visible. Medium of culture: DMEM FBS 10% and commercial NGF at a concentration of 50 ng/mL.

Growth in presence of our synthesized proNGF