Description

After multiple pivots, we strayed far from our original goal of Limnospira Infant Formula Technology (LIFT), which aimed to create an alternative additive for infant formula, to Limnospira-inspired Foundational Technologies (LiFT). In LiFT, we conceptualized accelerators and plan to test our genetic engineering tools across cyanobacteria strains that are better known for faster growth rates. Cyanobacterial engineering is no easy feat, as depicted by our team’s journey in selecting a host organism. In order to achieve the original purpose of the project, the techniques developed during this project must be applied to SAG 85.79; further research can be conducted regarding the viability of Argonaute and Cas12a and even possibly Cas9 usage in SAG 85.79.

CRISPR-Cas9 vs Cpf1

During the project's development, time constraints required us to prioritize the most promising option for success as our chosen accelerator. As a result, we decided to discontinue exploring the possibility of utilizing Cas9 as literature has shown it to be toxic in cyanobacteria.^[1] However, this does not imply that Cas9 is not a viable option. The same literature that noted its potential toxicity also suggested that Cas9 may simply be less effective in transformation rather than entirely unsuitable as transformed colonies were still seen; this suggests a possibility of potentially implementing Cas9 as an accelerator in other possible cyanobacteria strains.

Argonaute Optimizing

As highlighted in the Integrated Human Practices section, Dr. Susan Golden proposed that eliminating the Argonaute gene could enhance transformation efficiency. This suggestion led us to speculate that Argonaute activity in cyanobacteria may result in off-target effects. Although the mechanisms by which naturally occurring pAgos acquire their guide DNA (gDNA) are still unclear, we hypothesize that when non-BLACKBIRD optimized plasmids enter the cyanobacterial genome, the restriction-modification system may identify them as foreign and target them for cleavage. The resulting "scrap plasmid" fragments could then be recognized by Argonaute proteins, which may bind to the fragmented DNA, using it as gDNA. This interaction not only promotes the degradation of the initial plasmid but also extends to the degradation of other plasmids within the cell. Therefore, removing the Argonaute gene could improve transformation efficiency by eliminating its role as an additional defense mechanism. Optimizing the Argonaute accelerator could be another possible avenue for us to go into. However, there are many other avenues for improvement and growth that will bring us closer to reaching our goal of producing infant formula additives.

Ethical Concerns

From the outset, we stated that the goal of this project was to develop a more cost-effective alternative for infant formula manufacturing. In order to bridge the gap between proof of concept to product, it is important that we find an alternative method of transformant selection. Although using antibiotic selection markers is one of the most commonly used techniques to isolate transformants, it may not be preferable from a consumer perspective. To ensure consumer satisfaction and market viability, one must develop a strain devoid of antibiotic resistance. This is due to the very simple concept that these consumables may pass their drug resistance to other organisms upon consumption.^[2]

Entrepreneurship

Lastly, in order to achieve our original vision and iGEM’s mission of serving the underserved, a large area of improvement would be the optimization of the strain and medium. Historically, the Aztecs cultivated spirulina in concrete ponds, which grew abundantly and provided a nutritious food source. With the bioengineering techniques available nowadays it is not hard to see a future where strains and mediums can be optimized based on local environments and on what supplements are most needed. In our case, BG-11 and L. fusiformis are not ideal for mass production. Implementing a cheaper growing medium and optimizing growing conditions for various strains would significantly reduce costs and increase accessibility. One promising direction is exploring media with less mineral concentration which would more closely mimic natural environments, such as DISCOVR media. ^[3] DISCOVR media offers an economically advantageous alternative to BG-11 media. The optimization of essential elements needed for growth like in BG-11 media is a particularly important avenue; especially to underserved communities who would benefit the most from a cheaper alternative. Revitalizing this long-lost technique of cyanobacteria cultivation brings us closer to a future where nutritional supplements, and even formula milk, are more accessible and equitable. By harnessing the full potential of cyanobacteria, we can pave the way for more sustainable and widespread nutritional solutions.