During the progress of our project, it has become apparent that there are many paths that we could pursue in the future to further expand and improve on our project.
- Computer Metabolic Modeling: The goal would be to use computational models of metabolic pathways in closely related organisms in an attempt to identify further genes of interest in the fatty acid synthesis pathway. Ideally such genes would allow for the maximum accumulation of fatty acids in the N. crassa cells with the minimum impact on cell growth and division. In the end this would hopefully enable us to develop the most efficient strain possible for our bioreactor.
- Growth Media: Further research into various types of growth media would potentially allow a greater variety of wastes to be used in our bioreactors. Having a wide variety of potential wastes to use in our bioreactor is very important. Depending on the time of year, local geography, climate and location, different wastes might be available. By characterizing these wastes and N. crassa's ability to grow on them, we could create a more universal bioreactor, allowing consumers to get the greatest yields from their investments.
- Large Scale Bioreactor: The creation of a safe, efficient and energy positive large-scale community based bioreactor would be the pinnacle of our project. Such a bioreactor would not only allow a community to be a step closer to energy independence, but also a step closer to a more sustainable future. This local reactor could become an integral part of the local economy as well by perhaps even paying the locals to bring their waste to the plant and by providing a very valuable resource to the community.
Developing a new chassis has been a very exciting endeavour for our team. The possibilities are endless. That being said, the first set of things to do would involve further developing the foundation of this chassis. By developing this foundation to a greater extent, it would give large toolkit for future iGEM teams
- Promoters: The characterization and creation of parts out of a variety of N. crassa promoters would be a great asset to the N. crassa toolkit. N. crassa has many interesting and unique promoters. Such promoters include a copper inducible promoter and several blue light inducible promoters. There are also promoters related to the circadian rhythm found in N. crassa. It would also be important to develop a catalogue of promoters with varying degrees of expression. Not all genes need to be expressed and extremely high levels, and overexpression of certain genes could be deleterious to the modified cells.
- Terminators: A variety of terminators would be especially beneficial to those who wanted to modify more than one locus in N. crassa. The more copies of the same sequence that are present in a N. crassa cell, the more likely there is to be homologous recombination between those sites. If such an event occurred, it could result in chromosomal rearrangements and deletions.
- Resistance Markers: A wider selection of resistance markers would allow future iGEM teams to choose a resistance marker that fits their experiment more appropriately. Some antifungals might be less toxic than others and certain antifungals might be less expensive than others. Also one could do several genetic modifications in the same strain and ensure that the resulting isolated strains contained all the desired cassettes.
- Phenotypic Markers: There are a variety of phenotypic markers that would be a great addition to the N. Crassa toolkit. The most fundamental one would be the coding sequence for a fluorescent protein that displayed a consistent phenotype when adequately expressed whose intensity varied on gene expression. The reason that this would be of such great value is that it would allow one to easily determine if a gene was being expressed and in what levels. This would create a very simple and elegant method of testing promoters and terminators. One could even compare various promoters to determine their relative strengths.
- Auxotrophic strains: Currently, there are a wide variety of auxotrophic strains that have been very well characterized in N. crassa. Not only do auxotrophic strains allow for a higher level of biosafety (eg. It is difficult for the auxotrophic strain to survive in the wild), but it would also possibly allow for the development of genetically engineered N. Crassa strains without resistance markers. By creating cassettes with the complimentary genes, it would be possible to easily identify transformed strains without the use of antifungals, allowing for even safer transformation procedures and protocols.
- Cre-Lox Recombinase System: The development of a Cre-Lox recombinase system as a potential engineering tool would be very beneficial to future iGEM teams. By creating a functional Cre-Lox recombinase system it would be possible to completely remove antifungal resistance genes after genetic modification and antifungal selection. N. crassa is unique in that it can create heterokaryons; fused cells with different nuclei. One can do this by inoculating opposite sides of a petri dish with different strains of the same mating type. Where the two meet they will form heterokaryons. By surrounding the antifungal resistance genes in the cassette with Lox sequences, one is essentially creating a region that the Cre recombinase can splice out. One would create heterokaryons by allowing the genetically modified strain with the Lox flanked resistance gene to grow beside the strain containing Cre recombinase. If the strain with Cre recombinase were auxotrophic, it would be relatively easy to purify the new strain with the resistance gene excised. One would just need to replate the resulting cells on an minimal media (to inhibit the growth of the cells whose nuclei contain Cre recombinase) and replicate plate the resulting colonies on the antifungal containing medium. This would allow one to select the colonies whose antifungal resistance gene has been removed. By plating a couple generations on minimal media, one could ensure that no Cre containing heterokaryons still present, and that all one would have left are homokaryons without the antifungal resistance gene.
- Mating: The development of efficient mating protocols would allow the development of a system that has the precision and ease of genetic manipulation of the mus strain, but allows the creation of a genetically modified strain without the mus phenotype. To do this, one would do all their genetic manipulation in the mus strain. They would then mate their modified strain with a wildtype strain and select for the mus negative strains that contain the modified insert. This would work best if all manipulated genes were on the same chromosome, but not the one that the mus deletion was on.