Biological nitrogen fixation has served as an inspiration for iGEM teams to build their projects around for many years, and as long as no solution is found to the nitrogen crisis, we believe many more will contribute towards solving it. We conceived NitroBLAST as a foundational project: a way to start a line of research on this exciting and new possibility to engineer a biological nitrogen fixation system. There is so much left to do on the road to creating nitrogen-fixing eukaryote strains, and in the spirit of iGEM, we documented all we have learned from our research as well as interviewing numerous experts, to provide tools and guidance for future teams to pick up where we have left off.

UCYN-A is actively undergoing genome reduction as part of its evolution towards an organelle: the reason it cannot live independently is that essential proteins for its survival are no longer present in its genome, but are now encoded in B. bigelowii’s and then imported into UCYN-A. Using recently released proteomics data on B. bigelowii and UCYN-A, along with older genomics and transcriptomics data, we have identified a putative list of proteins that are imported into the organelle. This data provides a solid foundation for further research into which proteins are essential, as we suspect many are redundant. Identifying a list of essential host-encoded proteins is crucial to successfully transplanting UCYN-A into a new host.

We have also created a new transcriptome assembly of B. bigelowii based on raw data from previous studies, using improved algorithms. This allowed us to create a new predicted proteome. We are making all of our omics data available on request for future iGEM teams along with documentation.

You can read more here.

Based on previous studies and the proteomics data on B. bigelowii we identified a strongly preserved motif on the C-terminal end of B. bigelowii proteins that are imported to UCYN-A. This sequence was hypothesized to correspond to a transit peptide (UCYN-A transit peptide or uTP), responsible for localizing proteins to the organelle.

The characterization of the transit peptide is a crucial step in understanding the nitroplast protein import pathway in B. bigelowii. The part allows future teams to run experiments aiming to deliver proteins to UCYN-A, whether in its native host to investigate its behavior or within a different recipient cell, to create an environment where the nitroplast could survive if transplanted. This all has implications on advancing research towards engineering new nitroplast-containing, nitrogen-fixing eukaryote strains.

You can read more here.

Based on the previously identified transit peptide and the predicted B. bigelowii proteome, we attempted to find any proteins which interact with uTP and thus might play a role in the UCYN-A protein import system. To achieve this, we began developing a proteome-scale peptide-binding prediction tool, which, to our knowledge, does not yet exist.

Unfortunately, due to the strict time constraints in iGEM, we were not able to finish work on this tool, but we are making our existing code and data available to aid any future teams.

Read more on our Materials and Methods page.

To facilitate the study of UCYN-A, a protocol to isolate the organelle from B. bigelowii is required. This has been done before, relying on a Percoll gradient. We attempted to develop an alternative protocol for future research via cell sorting which should prove quicker and less laborious, for future teams to be able to use.

You can read more here

Over the course of our project, while we worked on our entrepreneurship case, we found a lot of entrepreneurship jargon. As students of a technical university, and early career starters, we were not familiar with a lot of these words. To make it easier for future iGEM teams working on entrepreneurship cases and potentially a start-up from their iGEM team, we compiled a list of words and their meanings relevant to entrepreneurship in an entrepreneurship dictionary. Through this we hope to help future iGEM teams that might encounter confusions similar to us.

B. bigelowii is an organism of great interest thanks to UCYN-A. This is because the nitroplast is something like an “evolutionary snapshot” of organellogenesis, and its capability to fix nitrogen makes B. bigelowii the only known nitrogen-fixing eukaryote. However, B. bigelowii is not a model organism, and as a coccolithophore with no biosafety class assigned by any institution, it can prove complicated to gain authorization to work with it. Meanwhile, many experiments on our roadmap for the future of nitroplast transplantation involve modifying B. bigelowii, so this presented a problem.

We have worked together with our department's biosafety officer to submit a formal request to the Dutch National Institute for Public Health and the Environment (RIVM) for B. bigelowii to be approved into list A1. Organisms in this list are considered equal to common model organisms like E. coli in terms of biosafety and so they can be genetically modified in BSL-1 labs, insofar as no hazardous sequences are involved. We believe this classification will facilitate future research on B. bigelowii by Dutch teams immensely and should make it easier for teams from other countries to get clearance on experiments as well.