Delivery of the Cas9 or TevCas9 System into Phaeodactylum tricornutum via Conjugation of Plasmids from a Bacterial Donor

Wang, Helen; Slattery, Samuel S.; Karas, Bogumil J.; Edgell, David R.

doi:10.21769/bioprotoc.2974

“…In previous studies, the non-mobilizable helper plasmid pTA-Mob was used to transfer destination plasmids from bacteria to recipient bacteria [35], algae [10,12,13], and yeast [9]. The pTA-Mob plasmid encodes the machinery required for conjugal transfer of plasmids that contain an origin of transfer (oriT) [35].…”

Section: Resultsmentioning

confidence: 99%

“…Trans-kingdom conjugation is observed in nature in the form of T-DNA transfer from Agrobacterium species to plants [2,3]. For bioengineering purposes, various bacterial donor species have been used to deliver DNA to eukaryotic recipients such as the yeast Saccharomyces cerevisiae [4,5,6,7,8,9], algal diatoms Phaeodactylum tricornutum and Thalassiosira pseudonana [9,10,11,12,13], and mammalian cells [14,15,16,17].…”

Section: Introductionmentioning

confidence: 99%

Trans-Kingdom Conjugation within Solid Media from Escherichia coli to Saccharomyces cerevisiae

Soltysiak

¹

,

Meaney

²

,

Hamadache

³

et al. 2019

IJMS

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Conjugation is a bacterial mechanism for DNA transfer from a donor cell to a wide range of recipients, including both prokaryotic and eukaryotic cells. In contrast to conventional DNA delivery techniques, such as electroporation and chemical transformation, conjugation eliminates the need for DNA extraction, thereby preventing DNA damage during isolation. While most established conjugation protocols allow for DNA transfer in liquid media or on a solid surface, we developed a procedure for conjugation within solid media. Such a protocol may expand conjugation as a tool for DNA transfer to species that require semi-solid or solid media for growth. Conjugation within solid media could also provide a more stable microenvironment in which the conjugative pilus can establish and maintain contact with recipient cells for the successful delivery of plasmid DNA. Furthermore, transfer in solid media may enhance the ability to transfer plasmids and chromosomes greater than 100 kbp. Using our optimized method, plasmids of varying sizes were tested for transfer from Escherichia coli to Saccharomyces cerevisiae. We demonstrated that there was no significant change in conjugation frequency when plasmid size increased from 56.5 to 138.6 kbp in length. Finally, we established an efficient PCR-based synthesis protocol to generate custom conjugative plasmids.

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“…Plasmids pPtGE34 or pPtGE35, containing no guide RNA or sgRNA.UMPS.1944, sgRNA.UMPS.1646, sgRNA.UMPS.157, sgRNA.UMPS.311 for the PtUMPS gene, or sgRNA.PRAPHCH.929 or sgRNA.PRAPHCH.120 for the PtPRA-PH/CH gene, were conjugated from E. coli to P. tricornutum and exconjugants were selected on zeocin-containing media, supplemented with uracil or histidine as appropriate 41 . Ten colonies from each conjugation were resuspended in TE buffer and flash frozen at − 80 °C followed by heating at 95 °C to lyse cells and extract genomic DNA.…”

Section: Methodsmentioning

confidence: 99%

Plasmid-based complementation of large deletions in Phaeodactylum tricornutum biosynthetic genes generated by Cas9 editing

Slattery

¹

,

Wang

²

,

Giguere

³

et al. 2020

Sci Rep

Self Cite

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The model diatom Phaeodactylum tricornutum is an attractive candidate for synthetic biology applications. Development of auxotrophic strains of P. tricornutum would provide alternative selective markers to commonly used antibiotic resistance genes. Here, using CRISPR/Cas9, we show successful editing of genes in the uracil, histidine, and tryptophan biosynthetic pathways. Nanopore long-read sequencing indicates that editing events are characterized by the occurrence of large deletions of up to ~ 2.7 kb centered on the editing site. The uracil and histidine-requiring phenotypes can be complemented by plasmid-based copies of the intact genes after curing of the Cas9-editing plasmid. Growth of uracil auxotrophs on media supplemented with 5-fluoroorotic acid and uracil results in loss of the complementing plasmid, providing a facile method for plasmid curing with potential applications in strain engineering and CRISPR editing. Metabolomic characterization of uracil auxotrophs revealed changes in cellular orotate concentrations consistent with partial or complete loss of orotate phosphoribosyltransferase activity. Our results expand the range of P. tricornutum auxotrophic strains and demonstrate that auxotrophic complementation markers provide a viable alternative to traditionally used antibiotic selection markers. Plasmid-based auxotrophic markers should expand the range of genome engineering applications and provide a means for biocontainment of engineered P. tricornutum strains.

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“…Plasmids pPtGE34 or pPtGE35, containing no guide RNA or sgRNA#1, sgRNA#2, sgRNA#3 or sgRNA#4 for the PtUMPS gene, or sgRNA#1 or sgRNA#2 for the PtPRA-PH/CH gene, were conjugated from E. coli to P. tricornutum and exconjugants were selected on Zeocin TM -containing media, supplemented with uracil or histidine as appropriate 34 . Ten colonies from each conjugation were resuspended in TE buffer and flash frozen at -80 • C followed by heating at 95 • C to lyse cells and extract genomic DNA.…”

Section: Generation Of Ptumps and Ptpra-ph/ch Knockouts Using Cas9 Anmentioning

confidence: 99%

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

Slattery

¹

,

Wang

²

,

Kocsis

³

et al. 2020

Preprint

Self Cite

0

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The model diatom Phaeodactylum tricornutum is an attractive candidate for synthetic biology applications. Development of auxotrophic strains of P. tricornutum would provide alternative selective markers to commonly used antibiotic resistance genes. Here, using CRISPR/Cas9, we show successful editing of genes in the uracil, histidine, and tryptophan biosynthetic pathways. Editing events are characterized by loss of heterozygosity and by the occurrence of large deletions of up to ∼2.7-kb centred on the editing site. The uracil and histidine-requiring phenotypes can be complemented by plasmid-based copies of the intact genes after curing of the Cas9-editing plasmid. Growth of uracil auxotrophs on media supplemented with 5-FOA and uracil results in loss of the complementing plasmid, providing a facile method for plasmid curing with potential applications in strain engineering and CRISPR editing. Metabolomic characterization of uracil auxotrophs revealed changes in cellular orotate concentrations consistent with partial or complete loss of orotate phosphoribosyl transferase activity in knockout strains. Our results expand the range of P. tricornutum auxotrophic strains and demonstrate that auxotrophic complementation markers provide a viable alternative to traditionally used antibiotic selection markers. Plasmid-based auxotrophic markers should expand the range of genome engineering applications and provide a means for biocontainment of engineered P. tricornutum strains. pathways of P. tricornutum and show for the first time that plasmid-based copies of the intact PtUMPS and PtPRA-PH/CH genes can complement the uracil-and histidine-requiring phenotypes, respectively. Individual auxotroph strains are characterized by loss of heterozygosity at the edited alleles, and Nanopore sequencing of the edited population reveals large, heterogeneous deletions up to ∼ 2.7 kb. The uracil and histidine auxotrophs and their respective complementation markers are a potential alternative to antibiotic-based selection of plasmids in P. tricornutum. Our results also suggest a simple methodology to cure plasmids from uracil auxotrophs to enable strain and genome engineering.

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Delivery of the Cas9 or TevCas9 System into Phaeodactylum tricornutum via Conjugation of Plasmids from a Bacterial Donor

Cited by 10 publications

References 11 publications

Trans-Kingdom Conjugation within Solid Media from Escherichia coli to Saccharomyces cerevisiae

Trans-Kingdom Conjugation within Solid Media from Escherichia coli to Saccharomyces cerevisiae

Plasmid-based complementation of large deletions in Phaeodactylum tricornutum biosynthetic genes generated by Cas9 editing

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

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