FIGHT INFECTIONS
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 (Figure 11). 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: Schematic representation of the RIP production cassette.
We ordered to gene synthesize our DNA constructs. Once we received the sequence encoding for this production cassette named Seq8 (461bp) in the commercial plasmid pEX-A258, we amplified it 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).
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.
Alignment of Sequencing Results then 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 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).
Figure 15: Measurement of GFP fluorescence from S. aureus biofilms cultivated with different IPTG induction concentrations of RIP peptide. Every measure was done eight times and the bars show the average fluorescence. CM= Culture Medium from the induced E. coli culture.. SL = Lysis Supernatant from the induced E. coli culture.
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 experiments several times, and the results were not always as concluding. This variability is very likely due to a bias due to the different approaches used for supernatant removal and washes. When using the flicking approach, we damaged our biofilm. Then, we removed planktonic cells by micropipette.
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.
Figure 16: Measurement of absorbance at 570 nm S. aureus biofilms cultivated with different IPTG induction concentrations of RIP peptide and stained with Crystal violet. Every measure was done eight times and the bars show the average fluorescence. CM= Culture Medium from the induced E. coli culture.. SL = Lysis Supernatant from the induced E. coli culture.
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) Then biofilms were either stained with Crystal Violet 0.1% and resuspended in acetic acid 30% or 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.
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).
Figure 18: S. aureus sensor device and RIP production cassette
We ordered to gene synthesize our DNA constructs. 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 we amplified it 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.
