Progress 07/01/21 to 10/22/22
Outputs
Target Audience:The primary objective of this project was to develop in house technology and knowledge in support of a successful breeding program. The following efforts were undertaken to engage community and international audiences through these efforts: Internships. Over the duration of the project, research directly engaged multiple interns from underrepresented groups in STEM in hands-on project planning, research, and data analysis. Internships offered at a local Kailua-Kona, HI school engaged high school students with culture care, germplasm collection efforts, and experimental design. Additional weekly literature review sessions transferred skill in reading and comprehending scientific publications. Further experiments in life cycle control were conducted by an Asian American intern seeking her bachelor's degree in computational biology. In this role, she assessed the effect of temperature, irradiance, and spectra on the tetrasporogenesis of Asparagopsis taxiformis.`` Further laboratory instruction has disseminated skills in RNA isolation, DNA cloning, and polymerase chain reaction (PCR) which has led to new developments of cutting edge technology in genomics studying Asparagopsis taxiformis. Women lead investor presentations. Globally, female representation in company leadership is low, with women representing only 15% of executive positions. Further, women-led companies receive less than 2% of all venture capital. To work against these statistics, we prioritized women-led presentations on STEM achievements to investor groups (VCs, angels, foundations). All presentations highlighted our SeadedTM platform, becoming an integral part of closing a Series A Funding round of $7 million. The work achieved under our USDA SBIR Phase I proposal funneled VC investment into a woman owned and operated company. Community outreach. Staff R&D Manager Daisy Stock and business development intern Kylie Tuitivuki gave a live-streamed presentation on emerging breeding technology in partnership with the Maui Nui Marine Resource Council (MNMRC). MNMRC is a nonprofit organization working for healthy coral reefs, clean ocean water, and abundant native fish for the islands of Maui County. The presentation reached hundreds of community members and provided context for the research undertaken at our facility, especially in regards to environmental impact. Shey Dorji (Research Assistant) and Alexia Akbay (CEO) attended a shoreline fisherman association meeting, the Makahanaloa Fishing Association, comprised of over 150+ members mostly of Native Hawaiian descent to understand how improved seedstock could assist in seaweed regeneration activities on Hawaii's shorelines. Alexia Akbay visited the Waikalua Fishpond in Kane'ohe and met with local students on their seaweed growing project, explaining the importance of establishing seed banks for other native species. Alexia continues to mentor University of Hawai'i, M?noa Masters Student, Seanna Correa-Garcia, on her seaweed cultivation thesis project. International outreach. Research completed by this program was an integral component of efforts shared by CEO Alexia Akbay at the Tahiti Blue Ocean Climate Initiative (TBOCI), connecting our efforts with 250+ scientists, innovators, policymakers, business and financial experts, government representatives, community leaders, and environmental and youth activists. The shared focus of sustainable food supplies connects TBOCI and the USDA in a mission to feed the world sustainably, one which our research efforts addresses through supporting low impact beef, dairy, and leather products. Industry outreach and networking. Symbrosia presented live research at the Ocean Exchange 2021 and the Animal AgTech Innovation Summit 2022. Commercialization Manager Eli Etzioni presented on breeding technology, establishment of Asparagopsis seed banks, and the economic and environmental opportunities they present to 200+ ocean technology industry veterans from around the world and 600+ delegates from the livestock industry. Furthermore, these presentations were live streamed to maximize the reach of the information. Internal upskilling workshops. Upskilling workshops hosted by Symbrosia provided opportunities for interns and employees of gender and ethnic minorities to gain real world experience in areas of technology including CAD and 3D printing, coding, analytics, systems fabrication, experimental design and project execution. Aforementioned literature reviews taught comprehension skills in scientific writing that would have otherwise remained inaccessible to the uninitiated, and as a result informed genomics efforts through methods review conducted by interns and employees. Facility tours. Symbrosia outreach yielded tours with interested community members. This effort resulted in educational tours with traditional limu practitioners or limu loea (seaweed masters) of the community. Other audiences included local political representatives (county and state members), marine biologists, livestock farmers, angel investors, and citizen scientists. Disseminating knowledge of our research goals is crucial to maintaining mutual respect with indiginous and local people, who have historically collected Asparagopsis taxiformis for culinary and cultural purposes. Changes/Problems:5.1 Change in Project Director A request dated 8/5/2021 updated the Project Director of this grant from Jonathan Simonds to Daisy Stock. Mr. Simonds held expertise in environmental engineering, process engineering, scaled culture modeling, and was responsible for the experimental setup and establishment of methods. Team member Daisy Stock was initially onboarded to the project in a support role until identified for leadership. While the transfer of PD responsibilities posed no deviations from the original proposal, onboarding of the incoming project director required time. 5.2 Request for No Cost Extension Request submitted on 12/21/21 requested a no cost extension to October 28, 2022 to complete the remaining tasks as outlined in the initial proposal. The extension was granted by the review office. This request was necessitated by the aforementioned change in project director, travel restrictions due to the COVID-19 pandemic for collections, biohazard and environmental concerns related to genomics approaches, in addition to challenges posed by the complex and unknown life cycle triggers of target species Asparagopsis taxiformis. 5.3 Environmental biohazard of RNA isolation reagent High quantities of polysaccharides and polyphenols associated with cell walls of macroalga have long caused difficulties in nucleic acid extraction. Polysaccharides and polyphenols co-precipitate with nucleic acids which creates interference with downstream PCR applications. Rhodophyta, the group of eukaryotic algae containing species Asparagopsis taxiformis, are especially high in these polluting carbohydrate molecules and special techniques are required for clean nucleic acid isolation from them. Common chemical reagents for polysaccharide rich samples include mixtures containing sodium azide and beta-mercaptoethanol: two highly toxic compounds which are highly harmful to aquatic life. Challenges were increased by the facility's proximity to the ocean and a thriving, but delicate, coral reef that could be greatly harmed by leaching of these chemicals. In light of a lack of hazardous waste disposal on the Island of Hawai'i and the negative environmental impact these chemicals threaten, we experienced a months delay while working with Thermo Fisher Scientific to source an environmentally responsible alternative reagent. As a result of this collaboration, Thermo Fisher Scientific has been spurred to begin development efforts for lower toxicity alternatives to the highly toxic sodium azide and beta-mercaptoethanol, both found in reagents for plant RNA isolation, due to the aforementioned associated risks. 5.4 Timeline changes due to COVID-19 travel restrictions While the initial proposal stated that we would travel prior to the start of the grant to collect the founding population of germplasm, COVID-19 travel restrictions postponed these travel dates and ultimately contributed to the necessity of a project extension. The interisland travel quarantine expired at 11:59 p.m. on June 14, 2021, shortly before the start of the grant award period, limiting time for getting a head start on germplasm collections. Additional concerns over the hospital worker shortages and congested roads influenced a decision to prioritize the health of local communities over a head start on the grant work proposed. This also resulted in the deliberate decision to exclude the most rural location with the least health care resources for residents, Moloka'i, from germplasm collection efforts. What opportunities for training and professional development has the project provided?Internal upskilling workshops. Upskilling workshops hosted by Symbrosia provided opportunities for interns and employees of gender and ethnic minorities to gain real world experience in areas of technology including CAD and 3D printing, coding, analytics, systems fabrication, experimental design and project execution. Aforementioned literature reviews taught comprehension skills in scientific writing that would have otherwise remained inaccessible to the uninitiated, and as a result informed genomics efforts through methods review conducted by interns and employees. Internships. Over the duration of the project, research directly engaged multiple interns from underrepresented groups in STEM in hands-on project planning, research, and data analysis. Internships offered at a local Kailua-Kona, HI school engaged high school students with culture care, germplasm collection efforts, and experimental design. Additional weekly literature review sessions transferred skill in reading and comprehending scientific publications. Further experiments in life cycle control were conducted by an Asian American intern seeking her bachelor's degree in computational biology. In this role, she assessed the effect of temperature, irradiance, and spectra on the tetrasporogenesis of Asparagopsis taxiformis.`` Further laboratory instruction has disseminated skills in RNA isolation, DNA cloning, and polymerase chain reaction (PCR) which has led to new developments of cutting edge technology in genomics studying Asparagopsis taxiformis. How have the results been disseminated to communities of interest?Community outreach. Staff R&D Manager Daisy Stock and business development intern Kylie Tuitivuki gave a live-streamed presentation on emerging breeding technology in partnership with the Maui Nui Marine Resource Council (MNMRC). MNMRC is a nonprofit organization working for healthy coral reefs, clean ocean water, and abundant native fish for the islands of Maui County. The presentation reached hundreds of community members and provided context for the research undertaken at our facility, especially in regards to environmental impact. Shey Dorji (Research Assistant) and Alexia Akbay (CEO) attended a shoreline fisherman association meeting, the Makahanaloa Fishing Association, comprised of over 150+ members mostly of Native Hawaiian descent to understand how improved seedstock could assist in seaweed regeneration activities on Hawaii's shorelines. Alexia Akbay visited the Waikalua Fishpond in Kane'ohe and met with local students on their seaweed growing project, explaining the importance of establishing seed banks for other native species. Alexia continues to mentor University of Hawai'i, M?noa Masters Student, Seanna Correa-Garcia, on her seaweed cultivation thesis project. International outreach. Research completed by this program was an integral component of efforts shared by CEO Alexia Akbay at the Tahiti Blue Ocean Climate Initiative (TBOCI), connecting our efforts with 250+ scientists, innovators, policymakers, business and financial experts, government representatives, community leaders, and environmental and youth activists. The shared focus of sustainable food supplies connects TBOCI and the USDA in a mission to feed the world sustainably, one which our research efforts addresses through supporting low impact beef, dairy, and leather products. Industry outreach and networking. Symbrosia presented live research at the Ocean Exchange 2021 and the Animal AgTech Innovation Summit 2022. Commercialization Manager Eli Etzioni presented on breeding technology, establishment of Asparagopsis seed banks, and the economic and environmental opportunities they present to 200+ ocean technology industry veterans from around the world and 600+ delegates from the livestock industry. Furthermore, these presentations were live streamed to maximize the reach of the information. Facility tours. Symbrosia outreach yielded tours with interested community members. This effort resulted in educational tours with traditional limu practitioners or limu loea (seaweed masters) of the community. Other audiences included local political representatives (county and state members), marine biologists, livestock farmers, angel investors, and citizen scientists. Disseminating knowledge of our research goals is crucial to maintaining mutual respect with indiginous and local people, who have historically collected Asparagopsis taxiformis for culinary and cultural purposes. What do you plan to do during the next reporting period to accomplish the goals?
Nothing Reported
Impacts
What was accomplished under these goals?
Task 1: Collection of tetrasporophyte founding population The selection of an initial sporophyte population from the wild population that strongly expresses a desired trait is essential to an emerging breeding program. Traits selected for commercial interest vary in the wild population, with any variation increased by the presence of three evolutionarily distinct lineages in Hawai'i. We were able to estimate variance in yield at 25°C based on preliminary collected data, and create a model that predicts the necessary sample size to build a strain library where the yield of the top ten percent of performers is equivalent to that of the population. Task 2: Line 1-2 evaluation and selection Increasing real time gene transcription of Asparagopsis cultures was divided into two main stages. In the first stage we explored correlation between expression in the MBB1 and MBB4 genes and key metabolite production. Although this correlating link has been suggested in Thapa et. al 2020, the quantitative nature of this relationship is yet to be proven. We tested and developed a protocol to achieve high quality mRNA via liquid nitrogen and Guanidine isolation. Isolated RNA was transcribed to cDNA and utilized as the template for quantitative PCR to determine the relative gene expression of the MBB1 and MBB4 genes. High quality amplification of the two coding genes of interest and the housekeeping gene allowed us to quantify the relative rates of gene folding. Higher rates of gene folding is indicative that the gene of interest transcribed at a higher quantity. By performing a linear regression of gene folding versus bromoform concentration we were able to establish the correlation between MBB1 and MBB4 gene production and bromoform production at the same point in time. In line with the results from Thapa et. al, MBB1 gene is transcribed at a much higher quantity than the MBB4 gene. Due to monetary and time constraints we were unable to test MBB2 and MBB3, which are the other genes present on the MBB cluster. An R value of 0.78 on the linear regression for MBB1 and bromoform demonstrated a moderately strong linear relationship between the genetic activity and the phenotypic metabolite expression. Correlation between MBB4 and tissue bromoform levels was much lower. However, by averaging gene folding values of MBB4 and MBB1, the linear correlation to bromoform content was elevated to an R value of 0.82. This would indicate that all four coding regions on the MBB gene cluster are likely working together to produce bromoform. Future work would revolve around conducting functional gene analysis with the all four MBB genes and a much larger sample size. Task 3: Line 3-6 evaluation and selection In our Phase I proposal, we assumed through normal distribution that with a sample set of 100 individuals from around the Hawaiian islands, we would have an 80% chance of finding an individual with a DGR over 29.3% at 25°C. Following our analyses, we found that there was not normal distribution in the daily growth rates which amplified the differences between high and low growers. Based on collections conducted across the islands, we determined that a growth rate greater than 36.87% is representative of the top 10% of growth rates in the population at 25 degrees celsius. Increase the specific growth rate for all temperatures 18°C - 32°C in a genetic line. In the screening across all target temperatures, the top 10% of our strains had daily growth rates of 25.85% or higher. Targets set in the initial proposal forecasted growth rates of 15.5% in 18C, 30.3% in 25C, and 15.5% in 32C, with 10.7% across all temperatures. Through the work detailed here and conducted as part of the Phase I research, we determined the 90th percentile for growth. In 18°C there were 23 strains with growth rates of 10.00% or higher. In 25°C there were 38 strains with daily growth rates of 36.87% or higher. In 32°C there were 11 strains with daily growth rates of 25.85% or higher. In all targets, results were exceeded, producing a strain library of high caliber parents. Table 2. Targeted quantitative outcomes per line. Trait Lines Target Achieved MBB gene expression MBB1 MBB1 + MBB4 70-fold transcription of MBB1 10-fold transcription of MBB4 8-fold transcription of MBB1 1-fold transcription of MBB4 0.82 linear correlation between transcription rate and bromoform content Yield by temperature Low temp (18°C) Mid temp (25°C) High temp (32°C) Temp resilient (18-32°C) 15.5% DGR 30.3% DGR 15.5% DGR 10.7% DGR for all temps 26.0% DGR 65.49% DGR 52.29% DGR 55.52% DGR for all temps Of the top 10% ofsuccessful high growth candidates, 1/10 have been successfully initiated into breeding by means of the GENESIS chamber, with 9/10 remaining unfruitful at the time of submission. The individual currently in breeding was in the 90th percentile for growth rate in 18°C, establishing the first parent for a unique cold tolerant, high performance cultivar. ? Task 4: Tetrasporangia formation Utilizing the custom built culture conditions manipulator called the GENESIS, a series of experiments were run in order to determine a commercially viable prescription of conditions for tetrasporangia formation. At this time there are no companies with the ability to spawn gametophytes from sporophytes using tetrasporangia release with commercial reliability.However, via a proprietary set of conditions in the GENESIS chamber, the R&D team achieved controlled tetrasporangia development and release. Task 5 and 6: Gametophyte maturation, initial cross The primary risk identified in the undertaking of this research was the breeding cycle not to be completed in the allotted time period. Prior to the initiation of the grant, Symbrosia researchers began experimentation with the lifecycle to increase likelihood of lifecycle completion. Initial successes from these efforts include a greater understanding and control of the sporophyte and gametophyte life stages. Long term establishment of gametophytes in lab culture was not achieved. Tetrasporophyte tissue that produced tetrasporangia in the Symbrosia GENESIS chamber was cultivated in similar conditions that induced sporing. Tetraspores were successfully released and cultivated in enriched media of several recipes to encourage gametophyte establishment. However, germinated gametophytes were stunted in growth and displayed an abnormal morphology independent of vessel, media, or lighting conditions. Hypotheses behind this abnormal growth are based in our developing knowledge of the wild Asparagopsis holobiont. It is suspected that through repeated propagation in sterile conditions the microbial community of the algae is diminished. To bridge existing gaps in lifecycle understanding, concurrent experiments were conducted using wild type A. taxiformis gametophytes. Cystocarps were collected from mature gametophytic thalli from a predetermined site hosting mature carposporophytes. Cystocarps were removed mechanically with forceps for further experimentation. Experiments in varied shock treatments and photoperiods developed methods for spore release from this life stage. Task 7: Select F1 individuals The first filial generation of plant offspring resulting from a cross made of genetically distinct parental types was not achieved in this time period. It is expected to be completed in the first quarter of 2023 based on current research traction and the development of in-house technologies. Following the success of sporing and spore release experiments, Symbrosia has established successful control methods over both distinct periods of spore release to aid in future breeding efforts.
Publications
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