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).

Alignment of Sequencing Results then confirmed that pSB1C3 contained Seq1 and Seq2, BBa_K2616000 .

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 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.

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

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

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.

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).

Alignment of Sequencing Results then 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 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).

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.

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)

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.

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

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.

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

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.

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.

Growth in presence of our synthesized proNGF