Wetlab results
Below follows the results for the wetlab section of our project.
Mutation PCR Results
After several attempts at mutating stop codons into the CHI92 and SP1 genes, the mutation PCR gave a band of the expected size: 3044bp. This can be seen in the figure below.
These gel images suggest that we obtained functioning PCR settings for both a single nucleotide substitution and for a full codon insertion. These settings can be seen in the table below.
| Step | Temperature (°C) | Time (s) | Other |
|---|---|---|---|
| Initial denaturation | 98 | 60 | |
| Denaturation | 98 | 10 | |
| Annealing for substitution | 60 | 30 | 32 cycles |
| Annealing for codon insertion | 68 | 30 | 32 cycles |
| Extension | 72 | 90 | 30s/kbp |
| Final extension | 72 | 300 | |
| Hold | 4 | inf |
It would be interesting to further improve these settings to obtain a better result with more of the correct plasmid and less undesirable bands like those in the figures above, but that was not possible within the timeframe of this project.
Transformation of Plasmids
After the transformation of the mutated plasmids into E. coli, cell colonies grew. This suggests that at least the antibiotic resistance part of our plasmid was taken up by them. The transformation of the CHS3 plasmid into S. cerevisiae also yielded colonies, suggesting that at the very least the URA3 gene of the backbone was taken up.
To verify this, all three plasmids were extracted from the colonies and sent for sequencing. This showed a nearly 90% match between the designed promoter-RBS-Yuab section of the plasmid and the corresponding part of the sequencing result. This highly suggests that our entire construct is intact in the E. coli. As for the CHS3 plasmid, the received sequence had more mutations in it. This is not surprising since the CHS3 plasmid seemed to be contaminated with several additional sequences prior to transformation. Due to a lack of time it was transformed into the yeast regardless.
These results signify the effectiveness of the protocols used, as well as the construction of the plasmids. However they also highlight the need for further investigation into the CHS3 plasmid.
The final transformation into B. subtilis did not yield any colonies. There are a number of factors which might have caused this. Since the sequencing result of that plasmid was good, it is assumed that there is nothing inherently wrong with the plasmid. One reason for the failure might simply be that the transformation protocol either wasn’t very effective or that it was not followed properly, either in the media preparation or in the actual transformation steps. Missteps in either of these could have caused the high cell mortality. Another reason could be that B. subtilis does not take up plasmids as readily as E. coli does. E. coli has been genetically modified in several published studies, but it’s possible that it has a higher learning curve than model organisms. A final reason might have been a simple lack of luck. At times, it is hard to weed out the specific reason that a method did not work.
Drylab results
The docking simulations showed that both chitinase-92 and SP1 are capable of binding chitin-related ligands with comparable binding energies, particularly for the closed-ring structures. The lowest free binding energy was observed for the chitinase-92 NAG trimer complex, with a free binding energy of -5.23 kcal/mol, indicating a strong and stable interaction. The open-ring ligand docks with chitinase-92 were roughly 1.5 kcal/mol higher in energy, suggesting slightly weaker but still favorable binding. SP1 demonstrated stronger predicted binding to chitobiose, consistent with previously reported experimental adhesion results, while chitinase-92 showed stable interactions across multiple ligands.
However, due to the low-confidence structural model of SP1, these results should be interpreted with caution. The predicted binding sites were mainly located within structured regions of the proteins, with chitinase-92 favoring the interface between its two dense domains. Although the planar β-sheet region of SP1 was not accessible under the rigid docking conditions, other favorable binding pockets were identified. Overall, the results support the potential of both binding domains to mediate chitin adhesion, while also emphasizing the need for experimental validation to confirm the modeled interactions. A more detailed description and interpretation of the modeling and docking analysis can be found on our model page.
Project outlook
While this project did not manage to test the adhesion of B. subtilis to modified S. cerevisiae, it has laid the groundwork for future research to pick up where we left off. If future efforts are made, it will be interesting to see if a modified transformation protocol for B. subtilis is necessary, and if it is possible to make it bind to chitin. It would also be interesting to see this combined with an increased Iturin A production, as was done by Yue H. et al. 1, to study if that and/or the adhesion affects the survival of the target cells. Our specific area aside, we are looking forward to following the development of using bacteria to deliver drugs and if adhesion to target cells will be a large part of those efforts. Maybe the adhesion could be adapted to including a receptor which could cause the release of apoptosis-triggering molecules on to the yeast, much like how the immune system battles cancers 2.
Further into the future, it would be interesting to see this applied to actual C. albicans cells, both in single cell and hyphal form, as well as in larger models in clinical trials.
Future work in the dry lab could expand on our results by investigating molecular docking with a more advanced fungal cell wall model, in which chitin structures are accurately represented within the overall architecture. Such a model would enable a more realistic assessment of the adhesion properties of CHI92 and SP1 to the fungal cell wall, both in the yeast and hyphal morphologies. Additionally, further simulations could explore alternative ligands or docking partners to better understand binding specificity and optimize interaction strength 3,4,5.
When modeling skin infections, it will be important to consider alternative infection models, since structural differences in hair follicles and epidermal layers between species can lead to varying infection behaviors among fungal pathogens such as C. albicans and Candida auris 6.
Exploring these antifungal therapies for animals is also highly relevant from a global health perspective. Such treatments could contribute to food security and help mitigate the spread of antimicrobial resistance, a growing issue linked to the extensive use of antibiotics in livestock. Furthermore, advanced interventions in animals could reduce the risk of major zoonotic diseases—such as salmonellosis, campylobacteriosis, and pathogenic E. coli infections that can be transmitted from animals to humans 7.
References
- Yue H, Zhong J, Li Z, Zhou J, Yang J, Wei H, et al. Optimization of iturin A production from Bacillus subtilis ZK-H2 in submerge fermentation by response surface methodology. 3 Biotech. 2021 Feb;11(2):36.
- Kristenson, L. NK cell recognition of malignant cells - a CRISPR approach to define novel mediators. Department of Medical Biochemistry and Cell Biology, Institute of Biomedicine, Sahlgrenska Academy, University of Gothenburg. 2025.
- Arkowitz RA, Bassilana M. Recent advances in understanding Candida albicans hyphal growth. F1000Res. 2019 May 21;8:F1000 Faculty Rev-700. doi: 10.12688/f1000research.18546.1. PMID: 31131089; PMCID: PMC6530606.
- Chaffin WL, López-Ribot JL, Casanova M, Gozalbo D, Martínez JP. Cell wall and secreted proteins of Candida albicans: identification, function, and expression. Microbiol Mol Biol Rev. 1998 Mar;62(1):130-80. doi: 10.1128/MMBR.62.1.130-180.1998. PMID: 9529890; PMCID: PMC98909.
- Chaffin WL. Candida albicans cell wall proteins. Microbiol Mol Biol Rev. 2008 Sep;72(3):495-544. doi: 10.1128/MMBR.00032-07. PMID: 18772287; PMCID: PMC2546859.
- Saskia Seiser, Hossein Arzani, Tanya Ayub, Trinh Phan-Canh, Clement Staud, Christof Worda, Karl Kuchler, Adelheid Elbe-Bürger, Native human and mouse skin infection models to study Candida auris-host interactions, Microbes and Infection , Volume 26, Issues 1–2, 2024, 105234, ISSN 1286-4579
- Anee, I.J., Alam, S., Begum, R.A. et al. The role of probiotics on animal health and nutrition. JoBAZ 82, 52 (2021).