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Geneious, Thomas Scientific, Integrated DNA, New England Bio, Aether Bio, and Dean of Students and Dean of Engineering at UCSC
Contact Us
1156 High St, CA 95060, US
Our solution to elevating costs of formula:
Limnospira Fusiformis
Limnospira fusiformis (SAG 85), commonly known as spirulina, is a cyanobacteria that we believe is the most ideal organism for cultivating alternative infant formula. L. fusiformis is packed with amino acids necessary for development such as histidine, leucine, methionine, tryptophan, and valine. [1] According to the USDA, spirulina provides at least some amount of all but 7 out of 32 key ingredients in commercial formula. Among these, spirulina is seriously deficient, but not entirely devoid, of 6. [2] Furthermore, it also contains phenolic acid, tocopherol, linoleic acid, and has a high protein content; all this makes it a suitable candidate as a milk substitute.
However
Met with the challenges of working in L. fusiformis, our team made the decision to focus on establishing genetically tractable systems in L. fusiformis. This led to our experimentation on well documented and established model cyanobacteria, UTEX 2973 (Synechococcus elongatus). Currently, there are no readily available cyanobacteria transformation techniques that could be done in under two weeks. By tackling the exigence of our inspiration, we aim to foster a more sustainable and innovative future for cyano-engineering.
Our pivot from L. fusiformis:
UTEX2973
UTEX3154
UTEX2973
PCC7942
UTEX 2973
Synechococcus elongatus, like L. fusiformis, is a polyploid cyanobacteria. The main difference lies in the high transformation efficiency, the rapid replication rate, and the extensive genetic characterization for this cyanobacteria; S. elongatus has a well-established system for genetic manipulation. We believe that existing techniques, that allow S. elongatus to be readily transformable, can be applied to transforming L. fusiformis. Much literature exists for reference and direct application to our project, such as the CyanoGate modular cloning tool kit (Addgene kit #1000000146). The contents of the kit are specifically designed for S. elongatus and contain broad host range parts for other cyanobacteria.
UTEX3154
Synechococcus sp., UTEX 3154, is a close relative of Synechococcus sp. PCC 11901.[4]
One of the major limitations of PCC 11901 is its dependence on vitamin B-12, which increases the cost of the growing media and reduces the strain's appeal.[5]
Fortunately, UTEX 3154, which was developed by selecting mutants from PCC 11901 that thrive in media lacking vitamin B-12, proved to be a suitable alternative.
We received UTEX 3154 as a gift from Max Shubert and quickly noticed its fast growth rate, characteristic of PCC 11901.
Coincidentally, PCC 11901 happened to be the organism used in the paper that we based our double plasmid CRISPR-Cas12a accelerator off[6] ,
highlighting its potential as an engineerable cyanobacterial strain. PCC11901 has a few other attractive characteristics such as having one of the fastest known doubling
times of cyanobacteria (about 2 hours), and being able to reach a high density (about 30g/L). [5]
PCC7942
Synechococcus elongatus, PCC 7942, was the first documented cyanobacteria strain that is able to be reliably transformed with exogenous DNA. PCC 7942 is an obligate photoautotroph with a genome of approximately 2.7 Mb long. It is one of the most researched strains and was also used to develop model systems for the exploration of the prokaryotic circadian rhythm. We acquired this strain from the Golden Lab at UCSD, and it is nearly identical to UTEX 2973 with only a 55 single nucleotide difference. Mainly, PCC 7942 has a repaired spot mutation in the PilA gene making the strain naturally competent.[3] Despite the small differences, this strain grows three times faster than UTEX 2973 and displays high flexibility in terms of growth conditions.
We experimented with three main accelerator designs:
Single Plasmid System,
Cas12a Double Plasmid,
and
Argonaute Single Plasmid system.
Through LiFT we aim to create a foundation for a tractable system in cyanobacteria engineering as a means to eventually create a reliable biotechnological chassis for future applications Argonautes
References
[1] B. W. Jester et al., "Development of spirulina for the manufacture and oral delivery of protein therapeutics," Nature Biotechnology, vol. 40, no. 6, pp. 956-964, Mar. 2022, doi: https://doi.org/10.1038/s41587-022-01249-7.
[2] "FoodData Central," Usda.gov, 2024. https://fdc.nal.usda.gov/fdc-app.html#/food-details/170495/nutrients (accessed Sep. 15, 2024).
[3]“Home - Synechococcus elongatus PCC 7942,” Doe.gov, 2024. https://genome.jgi.doe.gov/portal/synel/synel.home.html (accessed Sep. 29, 2024).
[4]L. Mills et al., “Development of a Biotechnology Platform for the Fast-Growing Cyanobacterium Synechococcus sp. PCC 11901,” Biomolecules, vol. 12, no. 7, pp. 872–872, Jun. 2022, doi: https://doi.org/10.3390/biom12070872.
[5]Artur Włodarczyk, Tiago Toscano Selão, B. Norling, and P. J. Nixon, “Newly discovered Synechococcus sp. PCC 11901 is a robust cyanobacterial strain for high biomass production,” Communications Biology, vol. 3, no. 1, May 2020, doi: https://doi.org/10.1038/s42003-020-0910-8.
[6]A. J. Victoria et al., “A toolbox to engineer the highly productive cyanobacterium Synechococcus sp. PCC 11901,” PLANT PHYSIOLOGY, May 2024, doi: https://doi.org/10.1093/plphys/kiae261.
[7]J. Zhang, T. Sun, W. Zhang, and L. Chen, “Identification of acidic stress-responsive genes and acid tolerance engineering in Synechococcus elongatus PCC 7942,” Applied Microbiology and Biotechnology, vol. 108, no. 1, Jan. 2024, doi: https://doi.org/10.1007/s00253-023-12984-5.