Team:Pasteur Paris/Results

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RECONNECT NERVES

NGF Secretion BBa_K2616000



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 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, 12 and 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.

Sequencing results, when aligned with our original construct using Geneious 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. In Figure 4, we show that 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. The final basepair identity is 100%.

proNGF characterization and purification



Our chassis is Escherichia 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 Biotecnología of Madrid, bearing HlyB and HlyD (Type I secretion system) sequences, in order to get a chance to secrete proNGF 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 a single step Ni-NTA affinity purification column. We eluted our protein using a gradient of imidazole-containing buffer and one peak was detected (fraction A6).

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 a single step Ni-NTA column was not conclusive. Many proteins were 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 slices around 20 to 35 kDa of the FPLC flow-through (lane 2, Figure 6) by LC/MS/MS mass spectrometry, to verify the presence of proNGF.

With the LC/MS/MS analysis, 14 coverage unique peptides corresponding to proNGF were found in all fractions. The sequence coverage represents 63%. Results of mass spectrometry analysis demonstrate the expression 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 peptide. However these results are also consistent with a mix of cleaved and uncleaved proNGF. The TEV protease, 34 kDa fused to export singal and 28 kDa cleaved from the export signal are found.

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

Analysis of Fraction 5 of the gel shows that our protein proNGF is present in a mix of cleaved and uncleaved polypeptide (Figure 8). Mass spectrometry spectrum of Peptide A, IDTACVCVLSR, from proNGF sequence and peptide B IISAAGSFDVKEER from fused HlyA export signal are 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 export signal.

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 export signal. Sequence has been annotated to match corresponding protein amino acid sequences : In orange His tagged proNGF, in red TEV protease cleaving site, in pink HlyA export signal.
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 Ni-NTA affinity column, although in fraction A6 we also identified His-tag bound proNGF. To test if the His-tag is accessible for binding to Ni-NTA, we've performed a batch purification using Ni-NTA 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(DE3) pLysS with 1 mM IPTG and flow through when partially denatured.

Native His-tagged proNGF was not retained on Ni-NTA beads. We believe that the N-terminal His-tag may be hidden in the protein fold. Consequently, we denatured with 2M urea before purifying on the beads. As seen in lane 8 even 2M urea could not improve the binding. We also tried with an 8M urea concentration, without better results.

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

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


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 their N-termini (Figure 11).

Figure 11: Schematic representation of the RIP production cassette.

We gene synthesized our DNA constructs commercially. Once we received the sequence encoding for this production cassette, named Seq8 (461 bp) in the commercial plasmid pEX-A258, we amplified it in competent E. coli DH5α. After bacterial culture and plasmid DNA extraction, we digested the 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 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.

Sequencing results, when aligned with our original construct using Geneious, 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 concentrations during exponential phase at an OD600nm at 37°C. A 1 mL aliquot was centrifuged and the pellet stored at -20°C.
After two hours of induction, we centrifuged and collected both supernatant and pellet separately.

Test of RIP effect on S. aureus biofilm formation

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), (kindly provided by Pr. 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 of incubation at 37°C, 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 the impact of RIP on biofilm formation of S. aureus. In yellow, S. aureus alone with different concentrations of IPTG. In blue, S. aureus in the presence of culture Medium from induced BL21(DE3) E. coli expressing RIP. In green, S. aureus in the presence of the cell lysate supernatant from induced BL21(DE3) E. coli expressing RIP. Every measurement was done eight times and the bars show the average fluorescence.

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 those experiments several times, and the results were not always as concluding. This variability is very likely due to a bias linked to the different approaches used for supernatant removal and washes. When using the flicking approach, we damaged the biofilms. Therefore, we removed planktonic cells by micropipeting. This variability is often encountered when using this protocol, even in Pr. Jean-Marc Ghigo's laboratory.

Crystal violet staining



Since fluorescence measurements were not satisfying enough, we tried to improve our methods for quantifying biofilm formation. Thus, we began staining biofilms by Crystal violet 0.1% and measuring absorbance at 570 nm. Again, the results were very heterogeneous between our different experiments, and between the different protocols.
We tried to compare our protocol within the Institut Pasteur, but also outside of it. This was the occasion to collaborate with another iGEM team, namely the team WPI Worcester, who was also working on biofilm disruption. We decided to exchange our protocols. The results of this comparative experiment are shown in Figure 16.

Figure 16: Measurement of the absorbance at 570 nm of S. aureus biofilms after 0.1% crystal violet staining. We compared the washing protocols of our team (in red) with the one of WPI Worcester team (in blue). All biofilms were cultivated with varying concentrations of cell lysate supernatant from a BL21(DE3) E. coli culture induced with 0.1 mM IPTG for RIP peptide production. LS = Lysis Supernatant from the induced BL21(DE3)E. coli culture. NI=Non Induced. Every measurement was done eight times and the bars show the average measured absorbance.

We show that our method gave lower biofilm retention than WPI Worcester's. However, even if we obtained higher retention values with theirs, we still met the same variability, as seen by the error bars. This may be related to the use of various solvents, namely ethanol and acetone in our method, and acetic acid in their case. Mechanically, we applied the same steps in our first approach. Since there was no improvement, we switched to pipetting and then finally back to full tray washing again. Both protocols can be found here.

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). Biofilms were then either stained with crystal violet 0.1% and resuspended in acetic acid 30% or directly resuspended in PBS 1X. Surprisingly, with this method, the 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.
Potential ideas for improvement would first be to better standardize starting amounts of biofilm cultures. Secondly, to find more gentle planktonic cells removal methods. Thirdly, better staining methods in order to get better absorbance readouts that can also take into account biofilm formation on the walls of the 96-wells plate and not only on its floor. Finally, the use of RIP peptides that have been processed through the export machinery and that would be cleaved from their export signal might have higher activities.

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 their N-termini (Figure 18).

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

We gene synthesized our DNA commercially by Eurofins-Genomics. We received this genetic construct in three parts that we called Seq5 (1422 bp), Seq6 (960 bp) and Seq7 (762 bp) in the commercial plasmid pEX-A258 which we amplified in competent E. coli DH5α.

After bacterial culture and plasmid DNA extraction, we digested the commercial vector with XbaI and BamHI for Seq5, MscI, and SphI for Seq6, and HindIII and SpeI for Seq7. 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 with 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 for a good ligation. Due to the lack of time, we were not able to redesign the overlaps for this construction.

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

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 E. coli DH5α. 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).

We proved that our vector contained the insert by DNA electrophoresis (Figure 19).

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

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.

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: 100%.

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.

Figure 21: Effect of different temperatures on the growth of Cryodeath kill-switch transformed BL21(DE3) pLysS 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.

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.

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