Proof of Concept

OVERVIEW

Our goal is to construct a “Bio-carrier with a Biofilm Breaker Enzyme”, an Escherichia. coli strain that produces, carries, and releases enzymes that inhibit and/or destroy biofilm to increase the efficacy of disinfectants in onsen facilities.

As a proof of concept, we created a model biofilm that mimics those that grow in onsen, then checked their effects on biofilms using crystal violet assays and confocal laser scanning microscopy.

1.laboratory biofilm model with E.coli cultured in native onsen water

In this experiment, we used four E. coli laboratory strains (W3110, JM109, TG1 and RP437), and measured their capabilities to form biofilm when cultured in several "Onsen media” (Watanoyu, Bandai, Shima, Ho-shi and Ikaho) by crystal violet assays. "Onsen media” were prepared from the original onsen source, then we added glucose and casamino acid as nutrients to grow bacteria. The results showed that RP437 formed the most amount of biofilm when grown in Ikaho and Ho-shi onsen media (Fig. 1). For this reason, we decided to use RP437 for further experiments. The details on the collected onsen water can be found on the Integrated Human Practice page, and the detailed method on the Lab Notes Page.

Fig. 1 Biofilm mass on 96-well polystyrene plates by four E.coli laboratory strains when cultured in several "Onsen media".

2.Inhibition of biofilm formation using conventional enzymes

To determine which enzymes inhibit biofilm formation in our laboratory biofilm model, we tested the following four commercial products enzymes: α-galactosidase, β-glucosidase, DNase I and Proteinase K. The effect of these enzymes on biofilm formation was estimated with crystal violet assays.

We found that the addition of α-galactosidase, DNase I and Proteinase K decrease biofilm mass, but not β-glucosidase. These enzymes could decrease the biofilm of RP437 when grown in the Ho-shi onsen medium, but not the Ikaho onsen medium, in a dose-dependent manner. It was also noted that Proteinase K is the most potent enzyme. However, it is known that Proteinase K and DNase I are toxic for E. coli hosts when overproduced since chromosomal DNA and some beneficial proteins can be degraded, respectively. For these reasons, we focused on α-galactosidase for overexpression study in our project (Fig. 2).

Fig. 2 Biofilm mass on 96-well polystyrene plates with the addition of each enzyme incubated in Ho-shi and Ikaho onsen medium accordingly.

3.Inhibition of biofilm using the recombinant E.coli α-galactosidase

We tested whether the recombinant α-galactosidase inhibits biofilm formation. We first tested it using crude lysate from E. coli over-expressing α-galactosidase (DH5α carrying pSB1C3/α-gal-RFP) and the control E. coli strain (DH5α carrying pSB1C3/RFP). When we used 15 μg of the enzyme contained in the crude lysate, biofilm mass was reduced to 62.6%. We also used the "partly-purified” enzyme. Biofilm mass was reduced to 34.8% when incubated with 678 μg of the enzyme. These results indicated that α-galactosidase inhibits biofilm formation (Fig. 3).

Fig. 3 Biofilm mass on 96-well polystyrene plates when incubating the medium with nothing added and medium with an enzyme added. Left: 15 μg of the lysate, Right: 678 μg of the "partly purified” enzyme.

4.Degradation of biofilm using the recombinant E. coli α-galactosidase

We estimated the effect of the α-galactosidase recombinant protein on the degradation ofE.coli biofilm. We grew the biofilm by culturing E. coli RP437 for 24 hours in a 96-well plate. Then, we added the "partly-purified” enzyme from the α-galactosidase-overexpressing E. coli strain (DH5α carrying pSB1C3/α-gal-RFP) and incubated it for another 24 hours. Biofilm mass was reduced to 68.4% when incubated with 678 μg of the enzyme compared to the biofilm without the enzyme (Fig. 4).To characterize the structure of biofilm, we observed it on a confocal laser scanning microscopy after SYTO-9 staining. The RP437 biofilm after treatment with the "partly-purified” enzyme (1.02 mg or 5.09 mg) was thin and less dense compared to the control (Fig. 5).

Fig. 4 Biofilm mass on 96-well polystyrene plates when incubating bacteria for 24 hours then added the enzyme and incubated for another 24 hours.

Fig. 5 Bacteria stained with Syto9 were imaged as a green fluorescent color on the microscopy using a 60 objective. Overlooking (A) and cross-sectional (B) images were acquired for each sample.Left: Control, Middle: Enzyme at 1.02 mg, Right: Enzyme at 5.09 mg .

5. Degradation of biofilm using the bioengineered "Bio-carrier with a Biofilm Breaker Enzyme" that overproduces α-galactosidase and along with SacB.

We next constructed a biofilm-degradation system according to our idea which utilizes the recombinant α-galactosidase. Initially, we thought that directly adding α-galactosidase-overproducing strain may be able to degrade microbial biofilm. However, two considerable issues arose.

α-galactosidase is a bacterial cytoplasmic enzyme, thus it cannot target the biofilm matrix in extracellular space.
α-galactosidase-overproducer itself may be a pollution source if used in onsen facilities.

To solve these issues, we bioengineered a "Suicide Biofilm Breaker” (SM10λ carrying pABB-CRS2/sacB, pSB1C3/α-gal-RFP). This strain is an E. coli strain that produces SacB along with α-galactosidase and can be lysed by adding sucrose. We expected α-galactosidase recombinant enzyme to be released when this strain is lysed and degrades biofilm.

We estimated the effect of the α-galactosidase recombinant protein from "Bio-carrier" when exposed to sucrose on an E. coli biofilm. We grew the biofilm by culturing E. coli RP437 for 24 hours in a 96-well plate. Then, we added the Bio-carrier and incubated for another 24 hours in the presence of sucrose. Biofilm mass was reduced to 41.7% (Fig. 6). We also observed the structure of biofilm on confocal-laser scanning microscopy. The biofilm was decreased significantly after adding a "Bio-carrier with a Biofilm Breaker Enzyme" (Fig. 7).

Fig. 6 Biofilm mass on 96-well polystyrene plates when incubating bacteria for 24 hours then added the Bio-carrier E.coli and incubated for another 24 hours.

Fig. 7 Bacteria stained with Syto9 were imaged as a green fluorescent color on the microscopy using a 60 objective. Overlooking (A) and cross-sectional (B) images were acquired for each sample.Left:control, Right: Bio-carrier.

CONCLUSION

These experiments indicated that the recombinant α-galactosidase produced by our Bio-carrier has the ability to degrade biofilm formed by E.coli RP437 in conditions similar to onsen.
Observation by confocal laser scanning microscopy has also shown visually that biofilm mass has indeed reduced. The bacteria are likely to be less aggregated, and more susceptible to disinfectants without the protection from biofilm.
The Addition of sucrose has caused lysis of the Bio-carrier, which is significant for not only in releasing the Biofilm Breaker Enzymes, but also in assuring safety in preventing the release into nature, as described in Proposed Implementation.

FURTHER CONSIDERATION

These experiments should be followed up by testing the optimal conditions such as concentrations of the bio-carrier and sucrose, culture time of the bio-carrier in bathtubs and circulating apparatus.
Additionally, creating a model bathtub and circulation pipes would help in observing how and where the biofilms form, and whether our Biofilm-carrier adhere effectively.
Finally, we must confirm if the efficacy of disinfectants has increased, and whether usage of disinfectants in the cleaning process is significantly reduced.

Team:Gunma/Proof Of Concept