After successfully locating two known binding sites from well-studied clusters in the literature, we decided we would narrow our search down to
Aspergillus species only for follow up experiments. However, there were still 11,000 possible options.
The results of our proof of concept experiment showed that, of the 55 promoters in the A. nidulans aflatoxin cluster,
only 16 of them contained sites for the motif. However, in the A. fumigatus gliotoxin cluster, of it’s 24 promoters,
14 of them contained the motif binding site. Though this is a small sample size, it indicates that a high number of sites is
essential for finding successful sites in the sequences. This also indicates that the number of sites in each sequence can vary significantly.
To account for this uncertainty in our unknown experimental clusters, we select the largest unique clusters in Aspergillus
PWM: GliZ
PWM: AflR
Twelve Aspergillus clusters ranging from 50 - 80 genes in size were selected. Because many of these clusters are not characterized or
annotated in the JGI database, the specific metabolites created by these clusters, and even the transcription factors, are not specifically identified.
These clusters are identified only by their JGI cluster ID number and the species that they belong to. For these clusters, we followed the same
workflow as before: Each promoter of the cluster was extracted from the genome assembly and all promoters were converted to the positive strand.
These promoters were compiled into a single FASTA file and input into motifomatic with the same default parameters as the two successful clusters.
The success of the subsequent binding sites was assessed by the E-value and the number of sites found.
Due to limited cluster annotations, none of the clusters had a single, clearly identified putative transcription factor. As a result,
candidate transcription factors were chosen, and recorded alongside their proposed binding site. The information for each discovery was collected in a table
(below) for future wet lab verification.
Contribution
As our contribution to future iGEM teams, we provide open access to our public GitHub(https://github.com/igemsoftware2020), which contains all of our software used to obtain our list of putative binding sites. For those interested in testing or using our software in the future, we have provided tutorials and instructions.
We have also made available our procedural handbook for any future teams wishing to work with Aspergillus niger. This should greatly reduce the initial research time required to put together an effective protocol for growing spores.
Lastly, we have made available our list of putative binding sites, along with potential transcription factors. Confirmation of the functionality of these sites is something we wish to proceed with, but there is no reason why, based on our protocols, other teams cannot do this themselves.
Any further contribution to the catalog of filamentous fungi parts is welcomed and encouraged!
