Validating a Promoter Library for Application in Plasmid-Based Diatom Genetic Engineering

Abstract

While diatoms are promising synthetic biology platforms, there currently exists a limited number of validated genetic regulatory parts available for genetic engineering. The standard method for diatom transformation, nonspecific introduction of DNA into chromosomes via biolistic particle bombardment, is low throughput and suffers from clonal variability and epigenetic effects. Recent developments in diatom engineering have demonstrated that autonomously replicating episomal plasmids serve as stable expression platforms for diverse gene expression technologies. These plasmids are delivered via bacterial conjugation and, when combined with modular DNA assembly technologies, provide a flexibility and speed not possible with biolistic-mediated strain generation. In order to expand the current toolbox for plasmid-based engineering in the diatom Phaeodactylum tricornutum, a conjugation-based forward genetics screen for promoter discovery was developed, and application to a diatom genomic DNA library defined 252 P. tricornutum promoter elements. From this library, 40 promoter/terminator pairs were delivered via conjugation on episomal plasmids, characterized in vivo, and ranked across 4 orders of magnitude difference in reporter gene expression levels.

Genome-wide mapping of autonomously replicating sequences in the marine diatomPhaeodactylum tricornutum

“…By adding an origin of transfer (oriT) to the plasmid design, plasmids of a relatively larger size (e.g., 24-94 kbp) than those that could be introduced by using electroporation can then be introduced via conjugation (Karas et al, 2015). One of the major advantages is that as the size of the plasmid can be increased, restrictions on plasmid design can be reduced (Garza et al, 2023).…”

“…Furthermore, the fact that plasmids are transferred to diatoms in the form of episomal DNA via conjugation distinguishes it from electroporation (Kassaw et al, 2022;Garza et al, 2023). The phenomenon where a transgene inserted into genomic DNA is silenced due to various mechanisms, such as chromatin modification or transcriptional regulation, is a clear limitation of electroporation (Neupert et al, 2020).…”

“…The phenomenon where a transgene inserted into genomic DNA is silenced due to various mechanisms, such as chromatin modification or transcriptional regulation, is a clear limitation of electroporation (Neupert et al, 2020). In contrast, the ability to introduce plasmids in the form of episomal DNA is a great advantage of conjugation when used to create diatom transformants (Garza et al, 2023).…”

“…During the initial studies when conjugation was attempted for diatom transformation, it was hoped that the plasmid could be stably maintained in the transformant as episomal DNA by including yeast CEN/ARS in the plasmid design (Karas et al, 2015;Diner et al, 2016). Previous studies have reported that yeast CEN/ARS provides stability to the episomal DNA introduced in diatom transformants (Garza et al, 2023), but it was revealed that the functionality provided by it had limitations in maintaining episomal DNA (Diner et al, 2017;Kumar et al, 2020).…”