Date of Award
Doctor of Philosophy (PhD)
Synechococcus elongatus UTEX 2973 is the fastest growing cyanobacterium discovered to date. Using water, carbon dioxide, and light alone, this organism can double in 1.5 hours under optimal conditions. The accelerated doubling exhibited by Synechococcus 2973 makes it a prime candidate to serve as a model photoautotrophic system. However, Synechococcus 2973 lacks one highly desirable feature: it cannot undergo natural transformation. This thesis seeks to engineer this capacity into this fast-growing system in order to create an organism that is both fast growing and naturally competent. Synechococcus 2973 is a unique platform because it is >99% genetically identical to another model cyanobacterium, Synechococcus elongatus PCC 7942, which is naturally transformable. However, this sister system grows much slower (doubling in 4 hours under optimal conditions). In this thesis, a CRISPR/Cas genome editing system was developed for cyanobacteria to allow polymorphic alleles from Synechococcus 7942 to be substituted into Synechococcus 2973 to introduce natural competence to the fast-growing strain. After developing this system, this strategy was used to identify the loci that give rise to the accelerated growth phenotype. Additionally, RNA-sequencing analysis was performed to identify transcriptome differences between Synechococcus 2973 and Synechococcus 7942. With this information in hand, further analysis was performed to identify specific polymorphic loci that give rise to differential natural competence between the two strains. This approach identified two genetic loci that are required to introduce natural competence into Synechococcus 2973: structural transformation pilus component pilN and circadian transcriptional master regulator rpaA. Unfortunately, this engineered strain grew at a reduced rate (doubling in >4 hours), therefore, a directed evolution strategy was used to select against slow growth and to ultimately generate the Synechococcus 2973-T strain. This strain is both fast-growing and naturally transformable and will be an asset for researchers seeking a photoautotrophic model system with these features.
Chair and Committee
Himadri B. Pakrasi
Joshua A. Blodgett, Michael G. Caparon, Susan K. Dutcher, Louis A. Sherman,
Wendt, Kristen Elizabeth, "Engineering Natural Competence into the Fast-growing Cyanobacterium Synechococcus elongatus UTEX 2973" (2020). Arts & Sciences Electronic Theses and Dissertations. 2353.