Plant

Cyanobacteria are ecologically significant due to their essential role in ecosystems and their promising applications in areas such as biofuels, bioremediation, and genetic engineering. In our project, LiFT (Limnospira-inspired Foundational Technologies), we initially selected Limnopira fusiformis, commonly known as spirulina as the basis for our accelerator systems. However, we ultimately shifted our focus to cyanobacterial strains Synechococcus elongatus UTEX 2973, and Synechococcus elongatus UTEX 3154. This transition presented significant challenges, as it required us to navigate the intricacies of these organisms’ biological systems while continuously optimizing conditions for our experiment.

While many iGEM teams opt for organisms like E. coli as their chassis due to ease of handling and time efficiency, a smaller fraction explore cyanobacterial systems. In our project, we sought to push the boundaries of genetic engineering by developing innovative techniques for cyanobacteria. Our focus was on applying these methods to Limnospira fusiformis and similar organisms, with the aim of expanding possibilities for improving our accelerator systems.

Desired Chassis

When choosing our host organism, we prioritized specific traits to address key issues in current infant formula production systems. Through our research, we discovered that the high cost of formula is largely driven by the addition of supplements and preservatives. To tackle this issue, we focused on selecting an organism with a fast growth rate and naturally rich vitamin content. This led us to explore cyanobacteria as a potential solution. Certain species of cyanobacteria have extremely fast doubling rates, for example, UTEX 3154 has a doubling time of 2 hours. Several cyanobacteria species have an abundance of essential vitamins and minerals necessary for baby formula ranging from but not limited to sodium, potassium, niacin, and vitamin K. ^[1]

Host Selection

Our project began in Limnospira Fusiformis (UTEX 2340)^[2] because of its nutritional value which made for an excellent host organism off of which to base infant formula. However, we soon realized that the time constraints of the project would not allow us to fulfill the ambitious goals we had set for ourselves. Due to the lack of time and literature on transforming UTEX 2340, we decided to switch to another strain of cyanobacteria.

Like UTEX 2340, Synechococcus Elongatus (UTEX 2973)^[3] is a polyploid cyanobacteria, but has been much more researched. Because of this, we believe that by conducting our project in this host, we can use it as a proof of concept and transfer the protocols to UTEX 2340. However, we were faced with another problem in that UTEX 2973 was backordered so we would not receive it for another month. To waste no time, we switched to another host that was still very similar to UTEX 2340 and UTEX 2973.

Finally we chose Synechococcus sp. (UTEX 3154)^[4] which was given to us by Max Schubert. UTEX 3154 is yet another polyploid cyanobacteria that shares the same family as UTEX 2973. The important part of choosing UTEX 3154 is that it has a much faster growth rate than the previous 2 strains and the paper on which we based our protocols used PCC 11901 which is the parent strain of UTEX 3154.^[5]

Our Approach

Our approach to enhancing genetically characterized systems relies on two endonuclease systems: Cas12a and Argonaute. The use of Cas12a to help with genetic engineering in cyanobacteria was initially characterized by Victoria et al (2024) in which they established the “Double HR Technique”. The architecture of the double plasmid system largely inspired our adaptation which features our Cas12a gene block. Using our computational tool BlackBirdCoOp, RM “scars” were removed from the sequence, and the gene block was codon-optimized for our host organism. The Argonaute system, which remains largely uncharacterized with respect to genetic modification, is the more novel aspect of our project. The plasmid architecture of the Ago system tests the variables of Chi Site dependency, length of guide DNA, implementation of Restriction Enzyme sites, and Ago toxicity.

Our Achievements

We have consistently achieved steady growth in our two cyanobacteria strains, UTEX 3154 and PCC 7942, as demonstrated by their growth curves shown below. We were able to successfully grow those two strains, as well as UTEX 3222, using BG-11 media liquid culture.

In parallel with developing these model cyanobacteria for transformation, we have made significant progress towards developing a system of plasmid constructs to accelerate their genetic engineering. One of our key accomplishments has been the successful codon optimization of inserts using the BlackBirdCoOp software. These optimized inserts were then incorporated into a tiered assembly system, utilizing parts from both the MoClo and CyanoGate toolkits. So far, we have successfully completed Golden Gate Assembly reactions for both Level 0 and Level 1 constructs.

We believe we have successfully integrated key components, including AquI A, B, and C sites, upstream and downstream homology arms, Cpf1, and GFP; this is soon to be confirmed through sequencing. Our next goal is to construct a functional Level T assembly, which will facilitate the accelerated transformation of UTEX 3154 and PCC 11901.