Part:BBa_K531003:Experience

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Applications of BBa_K531003 by iGEM Stockholm 2016

We received BBa_K531003 and decided to use it in our project called SMITe - Spider silk Mediated Infection Treatment. Esp was used as one of our 'combat proteins' used with the intention of degrading biofilms commonly found in chronic wounds. If you'd like to learn more about our use of Esp, have a look at our [http://2016.igem.org/Team:Stockholm/Results-esp Esp Results section]. We added to existing characterization of Esp in four distinct ways:

- Assessing expression in a E. coli

- Investigating a possible bacteriolytic activity of Esp

- Describing Esp's ability to disperse pre-existing S. aureus and P. aeruginosa biofilms

- Improving existing characterization of Esp's ability to inhibit biofilm formation

Assessing expression in E. coli with a T7 promoter

The sequence of this BioBrick had been directly isolated from S. epidermidis by the iGEM Team Grinnell in 2011 and was expressedin S. epidermidis. In order to test whether this BioBrick could be expressed in E. coli as well and find wider use and application, we transformed plasmid containing Esp behind a T7 promoter into BL21(DE3) cells and induced protein expression. In our set-up, expression of Esp was hard to confirm without specific antibodies. In Figure 1, wells 6 and 8 correspond to insoluble fractions of cell lysates after 1 mM IPTG induction for 4 hours. In both of those wells, a band around 27 kDa (indicated by the arrow) can be observed, which corresponds to the size reported by other researchers (Sugimoto et al, 2011). After conducting the protein induction and SDS-PAGE several times with varying conditions, we were unable to detect stronger expression. Since the expression system was confirmed to be working with other BioBricks using the same assembly with T7, we assume that Esp might not be strongly expressed in these conditions. Since the BioBrick consists of the sequence as it was isolated from S.epidermidis it is likely that adaptations to the sequence would be necessary in order to increase the expression.

Figure 1: SDS-PAGE displaying soluble and insolube fractions of cell lysates. (2) and (3) soluble fraction, 1mM IPTG induction, (4) soluble fraction, uninduced, (6) and (8) insoluble fraction, 1mM IPTG induction, (7) and (9) insoluble fraction, uninduced.

Investigating a possible bacteriolytic activity of Esp

Esp displays a variety of functions and is not abundantly well characterized. (Sugimoto et al, 2011) We wanted to investigate whether Esp displayed any kind of bacteriolytic activity on TOB1 E. coli. A modified Kirby-Bauer test was used in our labs where filter papers were soaked in crude cell lysates of BL21(DE3) cells transfected with the Esp expression construct. They were placed on agar plates with E. coli spread on them, incubated at 37°C and bacterial growth was assessed at different time points. Different samples of Esp did show a certain inhibition of bacterial growth after 24 hours but are not sufficient to make any conclusions with this data alone. It remains up to future iGEM teams to collect more data and confirm or reject the hypothesis of a bacteriolytic activity.

Figure 2: Kirby-Bauer Disk Diffusion Test. Black asterisk indicate halo formation by Esp samples, red and green asterisks correspond to the matching negative and positive controls, respectively. Halo formation was observed for two samples - soluble fractions of cell lysates obtained after induction with either 1mM or 0.5mM IPTG

Providing additional data regarding biofilm dispersal of S. aureus and newly characterize the dispersal of P. aeruginosa bilfiolms

Data was collected with a well established biofilm assay - bacteria were grown for 48 hours before addition of the cell lysates containing Esp. The activity of Esp was assessed by staining remaining biofilm with crystal violet after 8 additional hours of incubation. our results clearly indicate a strong, biofilm-dispersing activity by Esp against both S. aureus and P. aeruginosa. Application of 50 μl of cell lysate containing Esp resulted in a strong decrease of absorbance, indicating detachment and degradation of biofilm. The results for this data point were highly significant when assessed using Student's t-tests, with p-values of 5.37225E-08 and 9.7229E-08 for S. aureus and P. aeruginosa biofilms, respectively.

Figure 3: Biofilm assay displaying the ability of Esp (here referred to as EB) to disperse S. aureus biofilms

Figure 4: Biofilm assay displaying the ability of Esp (here referred to as EB) to disperse P. aeruginosa biofilms

Improving existing characterization of Esp's ability to inhibit biofilm formation

In this setup we aimed to test Esp's ability to inhibition biofilm inhibition rather than to disperse pre-existing biofilm. Due to the different stages a biofilm goes through, from initial detachment to maturation to finally detachment and dispersal, the distinction between those two functions is vital (Otto et al, 2008). We were able to show clearly that Esp was able to also inhibit the formation of biofilm after 27 hours of incubation with P. aeruginosa. This adds to existing data of the Team Grinell 2011, indicating that Esp can inhibitS. aureus biofilms.

Figure 5: Biofilm assay displaying the ability of Esp (here referred to as EB) to inhibit P. aeruginosa biofilms

Summary

As can be seen in more detail on our [http://2016.igem.org/Team:Stockholm/Results-esp Esp Results page], we have expressed Esp in E. coli, demonstrated its biofilm-inhibiting and biofilm-degrading activity on two different clinically relevant bacteria, S. aureus and P. aeruginosa. Additionally we took first steps towards assessing a possible bacteriolytic activity of Esp though further investigations are needed.

References

Sugimoto, S. et al. Cloning, expression and purification of extracellular serine protease Esp, a biofilm-degrading enzyme, from Staphylococcus epidermidis. J. Appl. Microbiol. 111, 1406–1415 (2011).

Otto, M. Staphylococcal biofilms. Curr. Top. Microbiol. Immunol. 322, 207–28 (2008).

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