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- 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.

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.

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.

We also quantified biofilm formation by Crystal violet coloration, and then measuring absorbance at 570 nm. Again, the results were very heterogeneous between our different experiments.

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.

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.

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.

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