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-alpha 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.

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.

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).

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.

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.

Once we received the sequences encoding for this production cassette (named constructions Seq1, size 1096 bp and Seq2, size 1153 bp) in commercial plasmids from gene synthesis, 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 (NheI and BamHI for Seq1, MscI and HindIII for Seq2), extracted the inserts from the gel, and ligated it 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 contained the insert by electrophoresis.

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

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.

Our chassis is Escherichia coli BL21(DE3) pLysS, a specific strain dedicated to produce high amounts of desired proteins under a T7 promoter. Thus, we co-transformed our bacteria with BBa_K2611600 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 on 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 checked the presence of proteins in the fractions by SDS-PAGE (Figure 5). The expected size of our protein is 27 kDa if the signal peptide has been cleaved and 33 kDa if it is still bound. Thus we analyzed by mass spectrometry the bands corresponding to these weights to check the presence of proNGF.

We analyzed the proteins of the gel by LC/MS/MS in order to check the presence of proNGF. Mass spectrometry analysis of the fraction 5 of the gel shows our protein proNGF is present but is still bound to its signal peptide HlyA. proNGF is detected in the flow-through which means it was not retained by the column. We hypothesized the His-tag was hidden inside the protein. Consequently, we did not yet manage to purify the produced 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.