Part:BBa_K2616001
RIP (RNAIII-inhibiting peptide) export to periplasm of E. coli
This part allows to produce RIP after IPTG induction, and export it to the periplasmic space of E. coli through Type II Secretion system. RIP stands for RNAIII inhibiting peptide, and is an heptapeptide which interferes with S. aureus quorum sensing. It is composed of a T7 promoter, followed by DsbA export signal peptide and RIP sequence, followed by a terminator. After this first terminator, the system is repeated using MalE export peptide rather than DsbA.
We decided to modify the Biobrick BBa_K237002 from iGEM SDU-Denmark 2009 Team. This Biobrick enables expression of RNAIII inhibiting peptide, we improved it by adding a secretion signal peptide to adress RIP to E. coli Type II Secretion System and optimizing it for our chassis E. coli BL21 (DE3) pLysS strain.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12INCOMPATIBLE WITH RFC[12]Illegal NheI site found at 336
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal AgeI site found at 236
- 1000COMPATIBLE WITH RFC[1000]
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 1).
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 2, 3).
Figure 2: 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 3: 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.
Sequencing results, when aligned with our original construct using Geneious, confirmed that pSB1C3 contained Seq8, Biobrick BBa_K2616001
Figure 4: 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 concentrations during exponential phase at an OD600nm 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.
Test of RIP effect on S. aureus biofilm formation
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).
Figure 5: Measurement of the impact of RIP on biofilm formation of S. aureus. In yellow, S. aureus alone with different concentrations of IPTG. In blue, S. aureus in the presence of culture Medium from induced BL21(DE3) E. coli expressing RIP. In green, S. aureus in the presence of the cell lysate supernatant from induced BL21(DE3) E. coli expressing RIP. Every measurement was done eight times and the bars show the average fluorescence.
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 those experiments several times, and the results were not always as concluding. This variability is very likely due to a bias linked to the different approaches used for supernatant removal and washes. When using the flicking approach, we damaged the biofilms. Therefore, we removed planktonic cells by micropipeting. This variability is often encountered when using this protocol, even in Pr. Jean-Marc Ghigo's laboratory.
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 6.
Figure 6: Measurement of the absorbance at 570 nm of S. aureus biofilms after 0.1% crystal violet staining. We compared the washing protocols of our team (in red) with the one of WPI Worcester team (in blue). All biofilms were cultivated with varying concentrations of cell lysate supernatant from a BL21(DE3) E. coli culture induced with 0.1 mM IPTG for RIP peptide production. LS = Lysis Supernatant from the induced BL21(DE3)E. coli culture. NI=Non Induced. Every measurement was done eight times and the bars show the average measured absorbance.
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[http://2018.igem.org/Team:Pasteur_Paris/Protocols/CellBio]
Biofilm PFA fixation before staining
<p>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 7). Biofilms were then either stained with crystal violet 0.1% and resuspended in acetic acid 30% or directly 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 7: Biofilm culture fixed with Bouin's solution in 96-well microtiter 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.
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.
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