Proof of Concept
Spore Generation
Given the pivotal importance within the project context of a consistent, abundant supply of fresh, stable spores, an experiment was designed to compare the efficacy of two different liquid media compositions in producing a large amount of spores in the shortest possible timeframe.
The first investigated media was a traditional Luria-Bertani (LB) nutrient broth, but diluted 1:20 with sterile water: said dilution has been shown to strongly accelerate the process of nutrient depletion within the media following culture inoculation, pushing cells in a stress state and facilitating sporulation. The lack of nutrients ensures also a relative stability of the resulting spores, since low germinant concentrations mean higher chances of spores remaining dormant. The second one was instead the Difco sporulation media, a more recent broth showing promising sporulation capabilities. While being much richer on the nutritional profile than diluted LB, this media is supplemented with trace quantities of compounds that have shown a significant importance in triggering sporulation, such as iron sulphate and manganese chloride.
Despite the most established spore-harvesting methods using agar plates, from which the spores are scraped off and processed, a liquid culturing approach was instead tried in this case, due to its superior potential for spore production upscaling and requirements for the directed evolution spore screening pipeline.
B.subtilis SCK6 strain (a modified version of wild type B.subtilis featuring higher plasmid transformation efficiency) were cultured overnight in 4ml regular LB broth (250rpm, 37℃) and backdiluted to OD600 0.2. Samples were inoculated again in plain LB and their OD600 monitored constantly until reaching 0.8 (exponential growth phase).
At this stage, samples were spun down (13000g for 1min), washed with 1XPBS and finally inoculated in 10ml diluted LB and Difco sporulation media respectively. Samples remained in incubation (250rpm, 37℃) for 5 days. Every 24h, 1ml of sample was retrieved, heat shocked (70℃ fro 20 minutes) to eliminate any vegetative cells and be left with spores only, and then washed two times with 1XPBS, twice with sterile water and finally resuspended in 100ul sterile water.
To assess the amount of spores produced, a colony forming unit (CFU) counting approach was chosen. Purified spores were plated on LB agar plates in a 106 dilution with respect to the original culture volume, and left incubating overnight at 37℃. In addition to this, when retrieving the culture sample for spore preparation, the culture itself was plated on the LB agar plates, this time in a 107 dilution with respect to the original culture volume. This was done to be able to calculate the spore to vegetative cells ratio for each media at each investigated timepoint.
Germination
The results ultimately show the superior sporulation efficieny of Difco over diluted LB, as well as demonstrate its faster operating scale. In diluted LB, nutrient depletion causes lower biomass growth, resulting in turn in a lower final amount of spores produced: interestingly, spore production over time is very consistent, suggesting a state of balance between the germination of the newly produced spores into vegetative cells and death of the older ones. In Difco instead, a massive spike is observed in the production of both spores and cells within the first 48h of incubation: as mentioned before, Difco is a nutrient rich media, favourable for copious vegetative cell growth, while the presence of trace elements forces the strain into sporulation, producing large amounts of spores. However, the balance observed in the diluted LB case is not found in this occasion: increased nutrient availability means increased germination of the big quantities of newly produced spores, which add to the existing vegetative cells and, after 48h, quickly deplete the remaining nutrient causing a sharp fall in the cell population (hence strongly lowering sporulation too). In light of this considerations, Difco-mediated sporulation was chosen as the standard method for spore production throughout the project, and the optimal spore-harvesting time identified at 48h.
Fungal Inhibition Assay
The core of our project hinges on the biocontrol capabilities of B subtilis and on the assumption that these are present soon after germination of the spore. With our project, we’ve successfully proven that B. subtilis 168 exhibits antifungal activity against the pathogen Rhizoctonia solani, both in bacteria and spore form. Moreover, in vitro testing with the beneficial fungus Trichoderma rossicum only highlighted limited transient effects in inhibition of fungal growth. Together, these results point towards the fact that spores are an efficient and resilient delivery mechanism for the biocontrol activity of B. subtilis.
We performed dual culture assays for both R. solani and T. rossicum to evaluate the impact of B. subtilis’ biocontrol properties on fungal growth. Briefly, a dual culture assay was set up by placing a fungal mycelial plug in the middle of a Potato Dextrose Agar plate (PDA). You can find the full protocol here. In turn, 10 μL of either B. subtilis cell suspension at four different ODs ( 0.2, 0.4, 0.8 and 1) or an equivalent concentration of spores (ie. same number of CFUs) were spotted 2cm away from the middle of the fungal plug. Plates were then incubated at 27℃ for 5 days and the radius of mycelial growth was measured using Fiji. Importantly, in the case of measuring inhibition for T. rossicum at five days, the fungus grew around B. subtilis spots. Thus, when making our calculation we measured the radii of mycelia starting from the agar plug edge to the edge of the plate, subtracting the distance from which there was no growth around B. subtilis spots.
Fungal Phytophatogen
Rhizoctonia solani is a fungal phytopathogen with a wide host range and diverse disease manifestations, including root rot, one of the first plant disorders we learnt about during our stakeholder research.
Given the availability at the Stanley Lab (Imperial College London) of a R. solani strain, we decided to measure the antifungal activity of B. subtilis and evaluate whether the same degree of fungicidal action would be achieved in an assay with spores instead of cells.
In the control group, strains grow radially from the middle, whereas in the treated group, mycelial growth appears severely slowed down, not even able to reach B. subtilis spots. Notably, a dark halo appeared around the fungal plug. During our stakeholder interviews, we had the opportunity to show this data with plant pathologists and experts at the World Vegetable Centre. According to their informed opinion, R. solani appeared under stress when exposed to B. subtilis, with the brownish halo being testimony of a last resource response R. solani employed to neutralize B. subtilis in what effectively became an environment hostile for fungal growth.
From a qualitative standpoint, R. solani could be noted as being severely inhibited by B. subtilis. This was not only clear thanks to the limited outward expansion of the mycelium but the color change induced as well, a stress response symptom. Quantitatively, we see that after 5 days OD 1 concentration of cells, results in the highest level of inhibition with 76.4% on average, whereas after 10 days growth has all but ceased with inhibition levels ranging from 87.9 to 90.9%. This pattern is consistent in assays done with spores in the place of cells, as can be seen by figure .
These preliminary results justify the choice of B. subtilis as the host strain for our project with spores as a robust and effective delivery mechanism, as the display of chitinases is likely to work synergistically with the natural antifungal activity of B. subtilis and hence boost efficacy of our treatment.
Plant growth promoting fungus
One of the main concerns we received from our stakeholders, let them be experts in plant disease management or farmers themselves, was in regards to the impact of our product on the soil microbiome, especially beneficial fungi. Although a complete picture can only be obtained through in field trials, we wanted to perform a preliminary evaluation to assess whether B. subtilis possesses an antagonistic relationship or antifungal activity against a key beneficial fungi. Plant growth promoting fungi such as Trichoderma rossicum, enhance nutrient uptake, hormone production and prime plants for stronger immune response against pathogen infection.
A dual culture assay was conducted in the lab with the ascomycete fungus T. rossicum. This was selected not only due to its role in protecting plant health, but also due to its fast growing rates compared to other soil microorganisms. A wild type strain was sourced from Dr. Stanley’s lab at the department of Bioengineering, Imperial College London.
In contrast to what experienced with the phytopathogen, no dark/brownish halo was observed in this case, suggesting lack of a stress response. Moreover, although some degree of inhibition is noticeable nearby bacterial spots, it must be noted that mycelia appears healthy and able to grow around B. subtilis, colonizing the rest of the plate. This would preliminary serve to indicate a tolerative ability of T. rossicum towards B. subtilis, an hypothesis again confirmed from experts at the World Vegetable Centre.
Statistical Difference between Inhibition of Different Fungi
We conducted a one-way ANOVA with Tukey’s post-hoc correction test to compare the mean inhibition of our experimental results for T. rossicum versus that of R. solani. All bar charts are shown as mean plus/minus SEM.
A statistically significant difference was found between the percent inhibition at every OD of B. subtilis spores inoculated. This data points towards the fact that the early application of fungicides in spore form is a strong delivery mechanism, different than wild type. Nevertheless, wt cells still exibith a statistically significant activity when exposed to a pathogen (R. solani) vs a beneficial fungus (T. Rossicum).
Directed Evolution Pipeline
The inability to obtain the germinant receptor’s knockout strain in a timely fashion within the timeframe of the project led to some significant complication in the successful testing of the screening pipeline.
One of the main problems encountered in vitro during preliminary experiment of the pipeline was indeed finding a minimal media formulation that would not contain any known spore germinants, found in the majority of culturing broth formulations, to avoid the triggering of unselective germination, but at the same time rich enough to support growth of spores actually germinated into vegetative cells, to allow for the positive selection step of the pipeline. With a full knockout strain, spores could have been suspended in common, efficient nutrient broths such as the Luria-Bertani, as even in the presence of germinants the spore would have been completely “bulletproof” and theoretically able to germinate only in the presence of the successfully edited germinant receptor.
Extensive experimental work was done to identify a minimal media with these specific requirements, with two different formulations being investigated: M9 and C minimal media. All media contained the same carbon source, glycerol, and were inoculated with purified spores of wild type B.subtilis 168.
For each media, three distinct tests were carried out, supplementing one batch of samples with L-alanine, to observe the change in OD600 profile upon spore germination and growth, another one with N-acetyl-glucosamine (NAG), to be absolutely sure that NAG is unable to trigger germination of B.subtilis spores even in the absence of a tailor-engineered germinant receptor, and a control without any additional supplements.
Due to highly reflective nature of the thick spore coat, OD600 has been observed to decrease upon germination, as more and more spores loose their protective coat turning into vegetative cells, only to then start going up again following the traditional steps of the bacterial growth curve (exponential phase followed by a plateau and a gradual decrease).
These features can be seen in an unmistakable way when inoculating the purified B.subtilis 168 spores into LB, a rich media naturally containing several germinants: OD600 can indeed initially be seen decreasing, marking germination, and subsequently shoot up again, with vegetative cells thriving in this nutrient-rich environment.
Part of these features can be seen when inoculating in M9: a decrease in OD600 can indeed only be observed in the L-alanine supplemented batch, as expected. However, the large growth of vegetative cells found in LB does not seem to happen in this type of media, likely too poor on nutrients essential for exponential cell growth. Interestingly, OD600 of the NAG supplemented batch shows a moderate increase around 24h from inoculation, which could be linked to increased sporulation activity due to nutrient scarceness by randomly germinated spores.
IN C media, instead, the situation is far more chaotic, with all batches apparently germinating regardless the supplementation of germinants and their OD600 plummeting to extremely low values. These observation could be used as an indication that the formulation of the C minimal media itself could contain some germinants of unknown origin and dynamics, and that its composition might not be suitable to sustain prolonged cell growth.
Germination
In order to investigate the process of bacterial germination, we contacted Lea Bernier, a PhD from Stanley’s lab here at Imperial. During her PhD, she developed a microfluidic chamber device designed to image spores and characterise germination dynamics.
Fascinated by the opportunity, we asked Lea to help us image B. subtilis spores we produced as they underwent germination in rich media. Notably, the strain imaged presented a CgeA-GFP fusion which enabled display of the fluorescent protein on the outer spore coat. Fluorescence was then harnessed track the spore coat over time. As a consequence, we successfully gathered data on germination dynamics in this setting. Moreover, we demonstrated that germination is not impaired with protein display; indeed, the timescale and doubling times of the germinated spore are in line with what reported in the literature.[1]
To conclude, this data proves that in a timescale of minutes (30min) we can have full germination of cells in response to the detection of a germinant, justifying the engineering of spore receptors for the recognition of fungal biomarkers to achieve timely deployment of a biocontrol agent.
We would like this opportunity to thank Lea for all the help and support with this experiments!
Synbicite’s PoC grant: our next (big) steps
Across the timescale of iGEM, we were actively involved in fundraising to be able to access the competition. As we pitched our idea, we started noticing that people were incredibly excited about Sporadicate. Harnessing the excitement surrounding iGEM we built an extensive global stakeholder network that proved to be invaluable in our idea development.
With the preliminary data and momentum we have gathered, we have demonstrated that:
- There is is ground for engineering B. subtilis spores into commercially relevant biofungicides
- London’s synthetic biology landscape is thriving and there are opportunities to translate proof-of-concept projects to real products serving our target customer base.
- We are addressing a key unmet need, and thus there is interest from both industry and organisations like WVC and the CGIAR which focus on improving crop protection around the globe
Building on the above, we successfully applied and were awarded the Synbicite Proof-of-concept grant, a prize of £50.000 aimed at the development of a commercial prototype for Sporadicate.
The use of a targeted spore system is a novel approach to treating fungal plant diseases that has the potential to disrupt the global fungicide market. Not only do we expect greater efficacy than traditional fungicides, we project our solution to be a more cost-effective option for consumers.
With financial commitment from a UK translational centre come responsibilities. This grant will represent a great opportunity to build upon our gold medal criteria work, addressing and overcoming its limitations. The 6 month timeline of the grant will enable us to make significant progress in the more ambitious areas of our project, namely the engineering of the germinant receptor. Our plan is to proceed with the testing of the chimera, as well as complete the TWIST order already prepared by our dry lab team for screening. Indeed, having secured a knockout strain will enable us to circumvent all the issues faced with minimal media formulations, enabling us to utilize our high-throughput screening system.
References
[1]:San-Lang, W., Shih, I.-L.,Wang, C.-H., Tseng, K.-C., Chang, W.-T., Twu, Y.-K., Ro, J.-J. & Wang,C.-L. (2002) Production of antifungal compounds from chitin by Bacillussubtilis. Enzyme and Microbial Technology. 31 (3), 321–328. doi:10.1016/S0141-0229(02)00130-8.
[2]: Tsror, L. (2010)Biology, Epidemiology and Management of Rhizoctonia solani on Potato. Journalof Phytopathology. 158 (10), 649–658. doi:10.1111/j.1439-0434.2010.01671.x.