The implementation potential for this project goes well beyond the scope of a single iGEM project. By expanding and diversifying the catalog of parts in filamentous fungi, we provide more opportunity for the discovery of new antibiotics and environmental toxins. Our software, though it has only been used to find fifteen putative binding sites so far, is capable of locating many more, and is not limited to fungi alone. Our experimental design was built with the intention of being public and reproducible so that other researchers may be able to test our results in their labs.
Antibiotics and Industrial Use
Antibiotic resistance is an increasingly troublesome issue, especially when new antibiotic production is expensive and slow [1]. By creating software tools to expand the catalog of transcriptional machinery in filamentous fungi, we are taking the first steps toward new chassis organisms. New chassis organisms mean diversified methods for discovering new antibiotics. Imagine the applications for pharmaceutical companies, biotech companies, etc. With more tools to harness the complex biological circuits of filamentous fungi, we also open the door to producing more antibiotics, which prove difficult to produce in simpler organisms. As an example, the extraordinarily common antibiotic, penicillin, was not successfully produced in yeast until 2017, because of the many required components native to penicillium host cells.[2]
“Filamentous fungi have an arsenal of biological functions and are often superior to bacterial and yeast based production systems, in terms of metabolic versatility, robustness, and secretory capacity.” [3]
Our project would also benefit biotech companies like Marrone Bioinnovations and Novozymes, which specialize in things like soil screening and enzyme production. We spoke with scientists from these companies, asking them what sort of things would benefit them in their protocols. One suggestion was a more effective version of the program antiSMASH, which essentially helps locate biosynthetic gene clusters. While we didn’t exactly invent a better antiSMASH, our software tools make locating motifs within clusters more feasible, which could assist the company in their workflow. The researchers from both Marrone Bioinnovations and Novozymes agreed that some predictive computational tool for the discovery of fungal genetic parts would benefit them in their work. This is essentially what we are building.
A scientist from Novozymes expressed concern about the necessity for new binding sites, when there are already existing, known promoters that can increase the production of enzymes in filamentous fungi. However, where our project's point of view differs from the industrial point of view, is that we aim to diversify the genetic toolkit, instead of optimizing a tool for production. So in that sense, our project is more basic science, and less industry-driven.
Experimentation
Because of the lab closures this summer, we were unable to execute our experimental protocol. So, we wrote our experimental protocol with the intention of it being easy to follow by any researcher interested in analyzing our computational results. We even wrote a procedural handbook and made computational tutorials to make it easier for these other users. We are also eager to characterize and confirm the function of our novel binding sites on our own. We are hoping to undertake the task in our TEAM lab on the UC Davis campus under the supervision of Dr. Facciotti, once labs reopen. Should our putative binding sites be proved functional, the next steps would be discovery of other regulatory machinery within each cluster, primarily the transcription factors themselves. Once we have this data, we would aim to provide it to the public, most likely through publication in a scientific journal.
Safety Challenges
While Aspergillus niger is a biosafety level 1 organism, it is a common contaminant of food and a pathogen of multiple plant species. Care must be taken to ensure that A. niger spores are not released into the lab. This can be prevented by working in fume hoods.
Assuming our project arrives at an eventual point of secondary metabolite production from BGCs, we may run into safety issues in UC Davis’ TEAM lab. The lab is built for BSL 2 projects. Suppose we use our newly found genetic tools to study some BSL3 organism. In this situation, we would not be able to continue, unless we procured a space for BSL3 or higher experiments. We would also need to acquire the proper training, as we are only trained up to BSL 2.
Specifics from our safety protocol can be found in the procedural handbook
Experimental Challenges
While Plasmid pCT74 is available on the Life Science Market website for purchase, p502 is available only on the Fungal Genetics Stock Center website. One can order by emailing a formal university purchase order to questions@fgsc.net. If p502 is out of stock, we would need to locate a different plasmid that contains the rtTA2-M2 gene. Fortunately, there are multiple plasmids listed in databases online that contain the rtTA2-M2 gene.
If no expression of sGFP is observed, even before the addition of doxycycline.
If no change in expression of sGFP is observed after the addition of doxycycline.
If no colonies are observed on selectable media.
References
[1] Spellberg, Brad. “The future of antibiotics.” Critical care (London, England) vol. 18,3 228. 27 Jun. 2014, doi:10.1186/cc13948
[2] Awan, Ali R., et al. “Biosynthesis of the Antibiotic Nonribosomal Peptide Penicillin in Baker’s Yeast.” Nature Communications, vol. 8, no. May, Nature Publishing Group, 2017, pp. 1–8, doi:10.1038/ncomms15202.
[3] Health, Yale Environmental, and New Haven. Biological Safety BSL3 Laboratory Manual. no. September, 2017.
