Proof of Concept
Summary
As a proof of concept for the ideal goal of a biofungicide rhizobacteria, we’ve used BL21(DE3) pSB1C3::Chi18h8 in multiple functional assays. We successfully demonstrated the extracellular secretion of chitinase through SDS-PAGE analysis of the culture supernatant and proved that antifungal effector secretion is possible through fusion with YebF. We performed co-culture antagonism assays against S. cerevisiae to simulate antagonism against FOC, but due to the slow propagation of yeast across agar, our results were mostly inconclusive. Future experiments will need to be conducted in order to verify the activity of the secreted Chi18h8.
For a comprehensive experimental overview of our project, please visit the Experiments page.
Chitinase Secretion
To determine the secretion of Chi18h8-YebF fusion protein into the extracellular medium, we ran SDS-PAGE with supernatants collected from the centrifuged culture medium and cell lysate. Successful secretion means that we should observe Chi18h8 in both the cell portion and the culture supernatant, thus SDS-PAGE analysis of cell and supernatant samples should both yield Chi18h8 bands at 46 kDa; the protein size was determined by cross-referencing between the Benchling AA Sequence “Biochemical Properties” viewer and Berini et al.’s paper on Chi18h8 production.
Results
The first round of SDS-PAGE was performed on the unconcentrated culture supernatant of BL21 pSB1C3::Chi18h8.
SDS-PAGE, we did not see clear bands but faint bands on the gel. We considered that the secreted proteins may be diluted in the large volume of supernatant and couldn't be determined.
As expected, the protein concentration was too low to present significant results; all loaded samples presented faint bands due to low protein quantity. Notably, however, there seem to be bands between the 35 and 48 kDa present throughout the gel wells. It was initially hypothesized to be the secreted chi18h8; however the protein is roughly 46 kDa in size, and the band locations are inconsistent. Due to the faint bands and questionable band location, results from this trial are not convincingly conclusive.
To resolve the low protein concentration causing the faint bands, we adjusted protocols and reperformed experiments. We re-performed protein expression, and the resultant culture supernatants were collected after 4 hours of protein production and concentrated to a final volume of 1.2 mL with molecular weight cut-off spin columns.; ~30ml of supernatant was concentrated into a final volume of ~1.2ml; 150ul of the concentrated supernatant was used for sample preparation, then 20ul of the treated sample was used for SDS-PAGE.
The culture supernatants collected from both 30 oC and 37 oC incubation showed clear bands near 48 kDa, which was also seen in samples from cell lysates. This confirms that Chi18h8 has indeed been secreted into the extracellular medium, and confirms the function of our Chi18h8 construct.
Co-culture Assay
As a proof of concept for our future engineered B. subtilis biofungicide, we thought to perform a co-culture antagonism assay, which cultures BL21 pSB1C3::Chi18h8 with fungus, to verify that the secreted Chi18h8 does, in fact, have antifungal activity.
To achieve this, we designed our experimental setups by referencing the works of Anith et al. Our protocol for dual culture antagonism assay is performed by plating bacteria as a 3 cm vertical line 2.5 cm away from the center of the fungal agar disk inoculation point. Because the co-culture was between fungus and bacteria, Potato Dextrose Agar(PDA) was chosen as the agar medium. Bacteria from overnight liquid culture were inoculated as linear streaks with a sterilized inoculation loop and incubated overnight.
In our co-culture antagonism demonstration, we avoided handling real FOC due to safety concerns. The alternative candidate is S. cerevisiae, which shares similarities in cell wall composition and structure to FOC and makes it likely a viable choice. Furthermore, S. cerevisiae is largely safe and easily accessible via store-bought instant yeast. This gives S. cerevisiae a significant advantage over experimenting with filamentous fungi, as we had no samples of the latter. These factors made S. cerevisiae an ideal choice for our preliminary demonstration of co-culture antagonism via chitinase secretion.
Here, we’ll simply refer S. cerevisiae as yeast.
Results
To evaluate the inhibitory effect of Chi18h8 on fungal growth, we grew a test plate of yeast against BL21 pSB1C3::BBa_J04450 (control) and BL21 pSB1C3::Chi18h8 (experimental group), and a control plate of only yeast.
Percent inhibition would be calculated using the formula:
Unfortunately, the propagation of yeast across PDA is slow, and at the 144-hour mark the yeast barely grew 1 cm from the border of the agar plug. The yeast thus has to opportunity to interact with the bacterial streaks. Furthermore, no visibly discernible difference between the control and test plates was observed.
To speed up the process of yeast interaction with bacteria, we did away with the agar plug and plated overnight yeast liquid culture within the rough boundaries of a circle of radius 2 cm. However, the growth of the yeast was again too slow, and conclusive results couldn’t be drawn regarding the inhibitory effects of BL21(DE3) pSB1C3::Chi18h8 against yeast.
Due to a lack of clear protocol regarding bacteria-fungus antagonism research, our experimental designs were very crude.
Conclusion
Using BL21(DE3) pSB1C3::Chi18h8 in place of our theorized B. subtilis chassis, we demonstrated the successful secretion of Chi18h8 via pSB1C3::Chi18h8. However, chitinase activity against yeast is inconclusive. Future experiments will need to be conducted to further investigate chitinase effectiveness as well as various other functionalities of our engineered systems.
Future Experiments
Chitinase Secretion
Because the theory of our biofungicidal rhizobacteria hinges on chitinase secretion, we hope to further confirm its secretion through western blotting. During construct design for BBa_K4137009 (the chitinase secretion construct), we made the design choice of including a 6xHis tag. This allows us to use anti-His antibodies to perform antibody binding on Chi18h8 to conclusively confirm proper secretion.
Chi18h8 secretion could then be quantified. We could then proceed by creating alternative constructs designs utilizing different promoters, RBS’s, and terminators; using secretion quantity data we could then finalize a design with the highest level of secretion.
Antagonism Co-culture
Our decision of using yeast for co-culture was one of many compromises: yeast is not a filamentous fungus, so it doesn't truly emulate the growth of FOC; the cell wall similarity between yeast and FOC was a hypothesis based on literature analysis, but was not proved; in literature characterizing Chi18h8, yeast was not analyzed for inhibition by Chi18h8.
Thus, for future plans, we’ll instead use fungal species demonstrated to be inhibited by Chi18h8; we plan to acquire samples of fungi by contacting the Bioresource Collection and Research Center(BCRC) based in Taiwan. To avoid our initial safety concerns of operating with FOC by using safer, BSL 1 Category F. oxysporum strains. The F. oxysporum species is divided into forma specialis that target different crops, but genetic similarity within the species remains high; it can therefore be assumed that cell wall structures will be highly similar, so co-culture antagonism results done using non-FOC F. oxysporum samples should be applicable to FOC.
CcdA-CcdB Co-expression Assay
To ensure that CcdA really does bind to CcdB to prevent toxicity, we will perform protein expression in transformed BL21(DE3) pSB1c3::CcdA-6xHis::CcdB-Myc::mleR. This assembled plasmid will contain the entirety of the Toxin-Antitoxin system; by characterizing protein production via SDS-PAGE, quantification, and western blot, we’ll be able to transfer the system to B. subtilis for experimentation using our intended chassis. Construct designs could then be finalized to fine-tune bacterial survival conditions to the malate concentration present in banana plant root exudates.
Experimental Design Reflection
During co-culture assay experimentation, we observed that our RFP control group did not appear visibly red. This was alarming to us, as this potentially suggests that either the bacteria are dead or protein expression is not occurring. Both possibilities contributing to the lack of apparent redness would result in the failure of our co-culture assay. Thus, we will carry forward by troubleshooting bacterial growth and protein expression behavior on PDA plates to engineer a successful experimental design capable of producing valid results.
References
Berini, F., Presti, I., Beltrametti, F., Pedroli, M., Vårum, K. M., Pollegioni, L., Sjöling, S., & Marinelli, F. (2017). Production and characterization of a novel antifungal chitinase identified by functional screening of a suppressive-soil metagenome. Microbial Cell Factories, 16(1). Link to Source
Anith, K. N., Nysanth, N. S., & Natarajan, C. (2021). Novel and rapid agar plate methods for in vitro assessment of bacterial biocontrol isolates’ antagonism against multiple fungal phytopathogens. Letters in Applied Microbiology, 73(2), 229–236. Link to Source