An Expanded Plasmid-Based Genetic Toolbox Enables Cas9 Genome Editing and Stable Maintenance of Synthetic Pathways in Phaeodactylum tricornutum

Abstract

With the completion of the genome sequence, and development of an efficient conjugation-based transformation system allowing the introduction of stable episomes, Phaeodactylum tricornutum has become an ideal platform for the study of diatom biology and synthetic biology applications. The development of plasmid-based genetic tools is the next step to improve manipulation of this species. Here, we report the identification of endogenous P. tricornutum promoters and terminators allowing selective expression of antibiotic resistance markers from stably replicating plasmids in P. tricornutum. Significantly, we developed a protocol for sequential conjugation of plasmids from Escherichia coli to P. tricornutum and demonstrated simultaneous replication of two plasmids in P. tricornutum. We developed a simple and robust conjugative system for Cas9 editing that yielded up to 60% editing efficiency of the urease gene. Finally, we constructed a plasmid encoding eight genes involved in vanillin biosynthesis that was propagated in P. tricornutum over four months with no evidence of rearrangements, with whole-plasmid sequencing indicating that the majority of mutations occurred after plasmid assembly and initial conjugation rather than during long-term propagation. The plasmid-based tools described here will facilitate investigation of the basic biology of P. tricornutum and enable synthetic biology applications.

Rapid method for generating designer algal mitochondrial genomes

“…Next, the protoplasting solution was removed and the plugs were incubated with 5 mL of Proteinase K solution (100 mM EDTA (pH 8.0), 0.2% sodium deoxycholate, 1% sodium lauryl sarcosine, and 1 mg mL -1 Proteinase K) for 24 h at 50°C. Then, the plugs were washed four times as follows: twice with 25 mL of wash buffer (20 6 mM Tris and 50 mM EDTA, pH 8.0) for 2 h each at room temperature (RT), once with 25 mL of wash buffer containing 1 mM PMSF for 2 h at RT, and a final wash with 25 mL of wash buffer for 2 h.…”

“…">IntroductionPressing challenges in agriculture, medicine, and energy can be addressed through the use of designer organisms with engineered traits. The promise of synthetic biology lies in the ability to 3 build, deliver, and install synthetic genomes and biosynthetic pathways in specialized hosts.Phaeodactylum tricornutum is a model diatom algal species that is an attractive candidate for synthetic biology applications [1][2][3][4][5][6]. For example, P. tricornutum is a popular candidate for biofuel production due to its natural propensity for lipid storage [7].…”

“…The availability of genome sequences allowed for the development of genetic tools and DNA delivery methods such as biolistic-mediated transformation [11,12], electroporation [13][14][15] and bacterial conjugation [16,17]. Additional tools for P. tricornutum include a method for cloning whole chromosomes in yeast and Escherichia coli [18], characterized centromeres for maintaining episomal DNA [19], and genome-editing technologies [6,[20][21][22]. We now have a powerful arsenal of tools for engineering algal nuclear genomes; however, tools for engineering and delivering organelle genomes are still lacking.There are several advantages of engineering organelle genomes and installing synthetic DNA in these compartments rather than the nucleus.…”

Cas9-generated auxotrophs ofPhaeodactylum tricornutumare characterized by small and large deletions that can be complemented by plasmid-based genes

“…One microalgae species of interest is the marine diatom Phaeodactylum tricornutum. A variety of plasmid-based genetic tools have been developed for P. tricornutum that facilitate basic molecular manipulations and expression of complex synthetic pathways [3][4][5][6] . We, and others, have developed plasmid-based and DNA-free CRISPR (clustered regularly interspaced palindromic repeats) reagents for targeted editing of the P. tricornutum chromosome using the Cas9 protein (CRISPR-associated protein 9) [6][7][8][9][10] .…”

“…A variety of plasmid-based genetic tools have been developed for P. tricornutum that facilitate basic molecular manipulations and expression of complex synthetic pathways [3][4][5][6] . We, and others, have developed plasmid-based and DNA-free CRISPR (clustered regularly interspaced palindromic repeats) reagents for targeted editing of the P. tricornutum chromosome using the Cas9 protein (CRISPR-associated protein 9) [6][7][8][9][10] . These plasmid-based tools and synthetic pathways are currently maintained by available antibiotic-based selections, including Zeocin TM , phleomycin, nourseothricin, and blasticidin-S and their respective resistance genes, Sh ble, nat, and bsr [11][12][13][14] .…”