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).

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).

Sequencing results, when aligned with our original construct using Geneious confirmed that pSB1C3 contained Seq1 and Seq2, BBa_K2616000 .

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).

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

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.

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.

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).

Sequencing results, when aligned with our original construct using Geneious, confirmed that pSB1C3 contained Seq8, Bba_K2616001.

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 OD_600nm 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.

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).

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.

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.

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).

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.

BBa_K2616002

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).

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%.

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.

Biocompatibility

Conductivity

Platinum wire

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

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.

Neuron culture

Imaging was performed in collaboration with the BioImagerie Photonique platform of the Institut Pasteur. Data are presented as MEAN ± SEM. Significance between 2 different groups was determined using an Ordinary one-way ANOVA test on the software Prism6 (GraphPad). (ns: non-significant, *: p<0.05, **: p<0.01, ***: p<0.001, ****: p<0.0001)

As an alternative to our recombinant proNGF for control experiments, we performed an in vitro neural primary culture with commercial NGF. For this, a pair of E18 Sprague Dawley cortexes were purchased from BrainBits.co.uk. We digested the tissue with manufacturer provided papain according to their protocol and seeded 40 000 dissociated neurons on our microfluidic chips with different conditions of culture for six days at 37°C, and 5% CO2.

On our two-chamber microfluidic devices, we seeded neurons only on one side. Fifteen chips were used in total. After six days, neurons are fixed with paraformaldehyde (PFA) 4% and stained with DAPI. For differentiated markers: MAP2 (coupled with Alexa Fluor 555), a cytoskeletal associated protein and Beta-III Tubulin (coupled with Alexa Fluor 488), one of the major components of microtubules and a neuron-specific marker were used.

We can see in Figure 11 that we had contaminations on many of our microfluidic chips, because we could not use antibiotic selection otherwise our bacteria would have suffered from it, and that most of our experiments could not be analyzed. However, no contaminations were apparent for eight of them. Two of these successful ones are displayed in Figure 12.

As we can see, we succeeded in growing the cells inside our device in the presence of Neurobasal, B27 and GlutaMAX medium. It is possible to see neurons passing through one chamber to the other in this experiment. Unfortunately, the PDMS of the microfluidic chips detached from the bottom of the glass culture dish, leading to the growth of cells not inside of the microchannel, but bellow them (Figure 13).

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.

We also tested the action of commercial NGF on our culture. Neurons were put in culture in the presence of commercial NGF at different concentrations: 50 ng/mL, 250 ng/mL, 500 ng/mL, 750 ng/mL and 900 ng/mL. The optimal concentration was determined by modeling of NGF diffusion inside the medium. It was possible to capture the cells passing through one of the chambers of the microfluidic chip to the other side during a time-lapsed using phase-contrast microscopy recorded for the first 48h of culture at the BioImagerie Photonique platform, proving that our device was working as expected (Video 1).

Growth in presence of our synthesized proNGF