Genetic Tools

Plasmids and Parts

  • Episomal Plasmids

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Any plasmid can be made into an episomal (extrachromosomal) replicating vector for P. tricornutum by:

 

  • Including the yeast CEN6-ARSH4-HIS3 sequence

  • Including a low GC content region similar to P. tricornutum centromeres

The most common way for episomal plasmids to be delivered into P. tricornutum is by bacterial conjugation. Therefore, plasmids delivered by this method require an origin of transfer sequence (oriT).

Commonly used episomal plasmids include:

Empty episomal vector for P. tricornutum. Contains CEN6-ARSH4-HIS3, ShBle selection marker (zeocin resistance gene), and selection and replication markers for E. coli and S. cerevisiae.

Designer diatom episomes delivered by bacterial conjugation. (2015).

https://doi.org/10.1038/ncomms7925

Empty episomal vector for P. tricornutum. Contains CEN6-ARSH4-HIS3, ShBle selection marker (zeocin resistance gene), and selection and replication markers for E. coli and S. cerevisiae.

Refinement of the Diatom Episome Maintenance Sequence and Improvement of Conjugation-Based DNA delivery Methods. (2016). https://doi.org/10.3389/fbioe.2016.00065

Empty episomal vector based on pPtPBR1, modified to include the nitrate reductase promoter for expression in P. tricornutum.

Unpublished. Based on: Molecular toolbox for studying diatom biology in Phaeodactylum tricornutum. (2007). https://doi.org/10.1016/j.gene.2007.05.022

Empty episomal vector based on pPtPBR1, modified to include the FcpB promoter for expression in P. tricornutum.

Unpublished. Based on: Molecular toolbox for studying diatom biology in Phaeodactylum tricornutum. (2007). https://doi.org/10.1016/j.gene.2007.05.022

Empty episomal vector for P. tricornutum. Contains CEN6-ARSH4-HIS3, nourseothricin selection marker, and selection and replication markers for E. coli, S. meliloti, and S. cerevisiae. Can be delivered to P. tricornutum via conjugation by E. coli or S. meliloti.

Designer Sinorhizobium meliloti strains and multi-functional vectors enable direct inter-kingdom DNA transfer. (2019). https://doi.org/10.1371/journal.pone.0206781

 
 
  • Integrative Plasmids

Integrative plasmids do not replicate extrachromosomally but rather integrate into the P. tricornutum genome randomly (under normal circumstances).

These plasmids are commonly delivered by microparticle bombardment/biolistic transformation.

Integrative vector for P. tricornutum. Contains a CAT selection marker (chloramphenicol resistance gene) under the control of the nitrate reductase promoter.

Transformation of diatom Phaeodactylum tricornutum by electroporation and establishment of inducible selection marker. (2012). https://doi.org/10.2144/000113881

Integrative vector for P. tricornutum based on cloning vector pSP73. Contains a ShBle selection marker (zeocin resistance gene) and a multiple cloning site for expression of a gene using the FcpA promoter.

Transformation of the diatom Phaeodactylum tricornutum (Bacillariophyceae) with a variety of selectable marker and reporter genes. (2000). https://doi.org/10.1046/j.1529-8817.2000.99164.x

Integrative vector for P. tricornutum based on pPha-T1, modified to contain the blasticidin-S deaminase selection marker (blasticidin-S resistance gene).

Blasticidin-S deaminase, a new selection marker for genetic transformation of the diatom Phaeodactylum tricornutum. (2018). https://doi.org/10.7717/peerj.5884

 
  • Promoters

Commonly used promoters for P. tricornutum:

Constitutive Promoters

An Expanded Plasmid-Based Genetic Toolbox Enables Cas9 Genome Editing and Stable Maintenance of Synthetic Pathways in Phaeodactylum tricornutum. (2018). https://doi.org/10.1021/acssynbio.7b00191

Development of gene expression system in a marine diatom using viral promoters of a wide variety of origin. (2008). https://doi.org/10.1111/j.1399-3054.2008.01089.x

Characterization of marine diatom-infecting virus promoters in the model diatom Phaeodactylum tricornutum. (2015). https://doi.org/10.1038/srep18708

An Expanded Plasmid-Based Genetic Toolbox Enables Cas9 Genome Editing and Stable Maintenance of Synthetic Pathways in Phaeodactylum tricornutum. (2018). https://doi.org/10.1021/acssynbio.7b00191

Development of a new constitutive expression system for the transformation of the diatom Phaeodactylum tricornutum. (2015). https://doi.org/10.1016/j.algal.2015.05.012

An Expanded Plasmid-Based Genetic Toolbox Enables Cas9 Genome Editing and Stable Maintenance of Synthetic Pathways in Phaeodactylum tricornutum. (2018). https://doi.org/10.1021/acssynbio.7b00191

Development of gene expression system in a marine diatom using viral promoters of a wide variety of origin. (2008). https://doi.org/10.1111/j.1399-3054.2008.01089.x

Development of gene expression system in a marine diatom using viral promoters of a wide variety of origin. (2008). https://doi.org/10.1111/j.1399-3054.2008.01089.x

Potentiation of concurrent expression of lipogenic genes by novel strong promoters in the oleaginous microalga Phaeodactylum tricornutum. (2019). https://doi.org/10.1002/bit.27110

High-efficiency promoter-driven coordinated regulation of multiple metabolic nodes elevates lipid accumulation in the model microalga Phaeodactylum tricornutum. (2018). https://doi.org/10.1186/s12934-018-0906-y

Potentiation of concurrent expression of lipogenic genes by novel strong promoters in the oleaginous microalga Phaeodactylum tricornutum. (2019). https://doi.org/10.1002/bit.27110\

An Expanded Plasmid-Based Genetic Toolbox Enables Cas9 Genome Editing and Stable Maintenance of Synthetic Pathways in Phaeodactylum tricornutum. (2018). https://doi.org/10.1021/acssynbio.7b00191

  • Tubulin Gamma Chain - Constitutive (Sequence)

An Expanded Plasmid-Based Genetic Toolbox Enables Cas9 Genome Editing and Stable Maintenance of Synthetic Pathways in Phaeodactylum tricornutum. (2018). https://doi.org/10.1021/acssynbio.7b00191

Development of endogenous promoters that drive high-level expression of introduced genes in the model diatom Phaeodactylum tricornutum. (2018). https://doi.org/10.1016/j.margen.2018.06.003

Inducible Promoters

Alkaline phosphatase promoter as an efficient driving element for exogenic recombinant in the marine diatom Phaeodactylum tricornutum. (2017). https://doi.org/10.1016/j.algal.2017.01.007

Characterization of iron-responsive promoters in the marine diatom Phaeodactylum tricornutum. (2014). https://doi.org/10.1016/j.margen.2014.01.005

Molecular toolbox for studying diatom biology in Phaeodactylum tricornutum. (2007). https://doi.org/10.1016/j.gene.2007.05.022

Molecular toolbox for studying diatom biology in Phaeodactylum tricornutum. (2007). https://doi.org/10.1016/j.gene.2007.05.022

An Expanded Plasmid-Based Genetic Toolbox Enables Cas9 Genome Editing and Stable Maintenance of Synthetic Pathways in Phaeodactylum tricornutum (2018). https://doi.org/10.1021/acssynbio.7b00191

An Expanded Plasmid-Based Genetic Toolbox Enables Cas9 Genome Editing and Stable Maintenance of Synthetic Pathways in Phaeodactylum tricornutum (2018). https://doi.org/10.1021/acssynbio.7b00191

Unpublished. Based on: Molecular toolbox for studying diatom biology in Phaeodactylum tricornutum. (2007). https://doi.org/10.1016/j.gene.2007.05.022

Characterization of iron-responsive promoters in the marine diatom Phaeodactylum tricornutum. (2014). https://doi.org/10.1016/j.margen.2014.01.005

Characterization of iron-responsive promoters in the marine diatom Phaeodactylum tricornutum. (2014). https://doi.org/10.1016/j.margen.2014.01.005

A CRISPR/Cas9 system adapted for gene editing in marine algae. (2016). https://doi.org/10.1038/srep24951

  • Nitrate Reductase/NR - Nitrate Inducible (Sequence)

​A fast and reliable strategy to generate TALEN-mediated gene knockouts in the diatom Phaeodactylum tricornutum. (2017). https://doi.org/10.1016/j.algal.2017.02.005

 
  • Terminators

Commonly used terminators for P. tricornutum:

An Expanded Plasmid-Based Genetic Toolbox Enables Cas9 Genome Editing and Stable Maintenance of Synthetic Pathways in Phaeodactylum tricornutum (2018). https://doi.org/10.1021/acssynbio.7b00191

An Expanded Plasmid-Based Genetic Toolbox Enables Cas9 Genome Editing and Stable Maintenance of Synthetic Pathways in Phaeodactylum tricornutum (2018). https://doi.org/10.1021/acssynbio.7b00191

Molecular toolbox for studying diatom biology in Phaeodactylum tricornutum. (2007). https://doi.org/10.1016/j.gene.2007.05.022

An Expanded Plasmid-Based Genetic Toolbox Enables Cas9 Genome Editing and Stable Maintenance of Synthetic Pathways in Phaeodactylum tricornutum (2018). https://doi.org/10.1021/acssynbio.7b00191

An Expanded Plasmid-Based Genetic Toolbox Enables Cas9 Genome Editing and Stable Maintenance of Synthetic Pathways in Phaeodactylum tricornutum (2018). https://doi.org/10.1021/acssynbio.7b00191

An Expanded Plasmid-Based Genetic Toolbox Enables Cas9 Genome Editing and Stable Maintenance of Synthetic Pathways in Phaeodactylum tricornutum (2018). https://doi.org/10.1021/acssynbio.7b00191

An Expanded Plasmid-Based Genetic Toolbox Enables Cas9 Genome Editing and Stable Maintenance of Synthetic Pathways in Phaeodactylum tricornutum (2018). https://doi.org/10.1021/acssynbio.7b00191

An Expanded Plasmid-Based Genetic Toolbox Enables Cas9 Genome Editing and Stable Maintenance of Synthetic Pathways in Phaeodactylum tricornutum (2018). https://doi.org/10.1021/acssynbio.7b00191

A CRISPR/Cas9 system adapted for gene editing in marine algae. (2016). https://doi.org/10.1038/srep24951

Unpublished. Based on: Molecular toolbox for studying diatom biology in Phaeodactylum tricornutum. (2007). https://doi.org/10.1016/j.gene.2007.05.022

An Expanded Plasmid-Based Genetic Toolbox Enables Cas9 Genome Editing and Stable Maintenance of Synthetic Pathways in Phaeodactylum tricornutum (2018). https://doi.org/10.1021/acssynbio.7b00191

 
 
  • Markers

Commonly used markers for P. tricornutum:

  • Blasticidin-S deaminase/Blasticidin Resistance Marker (Sequence)

Blasticidin-S deaminase, a new selection marker for genetic transformation of the diatom Phaeodactylum tricornutum. (2018). https://doi.org/10.7717/peerj.5884

  • CAT/Chloramphenicol Resistance Marker (Nulcear) (Request)

Transformation of diatom Phaeodactylum tricornutum by electroporation and establishment of inducible selection marker. (2012). https://doi.org/10.2144/000113881

  • NAT1/Nourseothricin Resistance Marker (Sequence)

Designer Sinorhizobium meliloti strains and multi-functional vectors enable direct inter-kingdom DNA transfer. (2019). https://doi.org/10.1371/journal.pone.0206781

  • ShBle/Zeocin Resistance Marker (Sequence)

Transformation of the diatom Phaeodactylum tricornutum (Bacillariophyceae) with a variety of selectable marker and reporter genes. (2001). https://doi.org/10.1046/j.1529-8817.2000.99164.x

  • UMPS/Positive Selection Uracil::Negative Selection 5-FOA (Sequence)

Cas9-generated auxotrophs of Phaeodactylum tricornutum are characterized by small and large deletions that can be complemented by plasmid-based genes. (2020). https://doi.org/10.1101/2020.04.12.038471

  • PRA-PH/CH/Positive Selection Histidine (Sequence)

Cas9-generated auxotrophs of Phaeodactylum tricornutum are characterized by small and large deletions that can be complemented by plasmid-based genes. (2020). https://doi.org/10.1101/2020.04.12.038471

  • PDS1/Norflurazon Bleaching Resistance (Sequence)

A novel endogenous selection marker for the diatom Phaeodactylum tricornutum based on a unique mutation in phytoene desaturase 1. (2019). https://doi.org/10.1038/s41598-019-44710-5

Regulation of the Expression of Intracellular β-Carbonic Anhydrase in Response to CO2 and Light in the Marine Diatom Phaeodactylum tricornutum. (2005). https://doi.org/10.1104/pp.105.065185

Development of a new constitutive expression system for the transformation of the diatom Phaeodactylum tricornutum. (2015). https://doi.org/10.1016/j.algal.2015.05.012

  • EGFP/Green Fluorescence Protein (Sequence)

Transformation of the diatom Phaeodactylum tricornutum (Bacillariophyceae) with a variety of selectable marker and reporter genes. (2001). https://doi.org/10.1046/j.1529-8817.2000.99164.x

  • EYFP/Yellow Fluorescence Protein (Sequence)

A redox-regulated type III metacaspase controls cell death in a marine diatom. (2018). https://doi.org/10.1101/444109

  • ECFP/Cyan Fluorescence Protein (Sequence)

Molecular toolbox for studying diatom biology in Phaeodactylum tricornutum. (2007). https://doi.org/10.1016/j.gene.2007.05.022

  • Tags and Introns

Tags

High-efficiency promoter-driven coordinated regulation of multiple metabolic nodes elevates lipid accumulation in the model microalga Phaeodactylum tricornutum. (2018). https://dx.doi.org/10.1186%2Fs12934-018-0906-y

Molecular toolbox for studying diatom biology in Phaeodactylum tricornutum. (2007). https://doi.org/10.1016/j.gene.2007.05.022

  • Omega Leader Sequence (Transcription Increase) (Sequence)

High-efficiency promoter-driven coordinated regulation of multiple metabolic nodes elevates lipid accumulation in the model microalga Phaeodactylum tricornutum. (2018). https://dx.doi.org/10.1186%2Fs12934-018-0906-y

Introns

An Expanded Plasmid-Based Genetic Toolbox Enables Cas9 Genome Editing and Stable Maintenance of Synthetic Pathways in Phaeodactylum tricornutum (2018). https://doi.org/10.1021/acssynbio.7b00191

An Expanded Plasmid-Based Genetic Toolbox Enables Cas9 Genome Editing and Stable Maintenance of Synthetic Pathways in Phaeodactylum tricornutum (2018). https://doi.org/10.1021/acssynbio.7b00191

An Expanded Plasmid-Based Genetic Toolbox Enables Cas9 Genome Editing and Stable Maintenance of Synthetic Pathways in Phaeodactylum tricornutum (2018). https://doi.org/10.1021/acssynbio.7b00191

An Expanded Plasmid-Based Genetic Toolbox Enables Cas9 Genome Editing and Stable Maintenance of Synthetic Pathways in Phaeodactylum tricornutum (2018). https://doi.org/10.1021/acssynbio.7b00191

 
  • Organellar Tools

These sequences are for expression and engineering in the chloroplast of P. tricornutum.

Construction of Novel Chloroplast Expression Vector and Development of an Efficient Transformation System for the Diatom Phaeodactylum tricornutum. (2014). https://doi.org/10.1007/s10126-014-9570-3

Construction of Novel Chloroplast Expression Vector and Development of an Efficient Transformation System for the Diatom Phaeodactylum tricornutum. (2014). https://doi.org/10.1007/s10126-014-9570-3

***Please note, Myc tags are typically the amino acid sequence EQKLISEEDL, however the provided sequence from the paper is missing the codon for I. The sequence from the paper is provided in the link above.

Construction of Novel Chloroplast Expression Vector and Development of an Efficient Transformation System for the Diatom Phaeodactylum tricornutum. (2014). https://doi.org/10.1007/s10126-014-9570-3

  • CAT Organellar Selection Marker (Chloramphenicol) (Sequence)

Construction of Novel Chloroplast Expression Vector and Development of an Efficient Transformation System for the Diatom Phaeodactylum tricornutum. (2014). https://doi.org/10.1007/s10126-014-9570-3

The following are plasmids designed for integration into the chloroplast genome:

Construction of Novel Chloroplast Expression Vector and Development of an Efficient Transformation System for the Diatom Phaeodactylum tricornutum. (2014). https://doi.org/10.1007/s10126-014-9570-3

Construction of Novel Chloroplast Expression Vector and Development of an Efficient Transformation System for the Diatom Phaeodactylum tricornutum. (2014). https://doi.org/10.1007/s10126-014-9570-3

Construction of Novel Chloroplast Expression Vector and Development of an Efficient Transformation System for the Diatom Phaeodactylum tricornutum. (2014). https://doi.org/10.1007/s10126-014-9570-3

 
 
 

Editing and RNAi

  • CRISPR/Cas9

CRISPR/Cas9 has been effectively used to edit and knockout targets in P. tricornutum. Single and double gene knockouts have been demonstrated with high efficiency.

Delivery of episomal vectors by bacterial conjugation can be used to introduce and express Cas9 and sgRNA inside of P. tricornutum and be lost by a subsequent curing step.

Biolistic transformation can be used to introduce either introduce plasmids to express Cas9 and sgRNA or CRISPR/Cas9 RNP complexes directly in a transgene-free manner. 

Both methods have benefits and disadvantages. In a head-to-head comparison, editing using an episomal system tended to require a slightly longer time requirement for the editing process, while the biolistic approach could result in issues due to accidental, random integration of plasmids.

Constructs for both methods to perform CRISPR/Cas9 editing in P. tricornutum are available on Addgene using links below:

Expression of the sgRNA and Cas9 on a non-episomal vector.

Expression of the sgRNA and Cas9 on a curable episomal vector.

Expression of the sgRNA and TevCas9 on a curable episomal vector.

To get started, here are some publications that have developed easy to use methods for designing your sgRNA construct, assembly of the final plasmid for transformation, transformation itself, and screening:

  • Episomal CRISPR/Cas9 Pipeline

An Expanded Plasmid-Based Genetic Toolbox Enables Cas9 Genome Editing and Stable Maintenance of Synthetic Pathways in Phaeodactylum tricornutum. (2018). https://doi.org/10.1021/acssynbio.7b00191

Cas9-generated auxotrophs of Phaeodactylum tricornutum are characterized by small and large deletions that can be complemented by plasmid-based genes. (2020). https://doi.org/10.1101/2020.04.12.038471

Delivery of the Cas9 or TevCas9 System into Phaeodactylum tricornutum via Conjugation of Plasmids from a Bacterial Donor. (2018). https://doi.org/10.21769/BioProtoc.2974

  • Biolistic CRISPR/Cas9 RNP Pipeline

One-step generation of multiple gene knock-outs in the diatom Phaeodactylum tricornutum by DNA-free genome editing. (2018). https://doi.org/10.1038/s41467-018-06378-9

  • Non-Episomal CRISPR/Cas9 Pipeline

Assessment of genomic changes in a CRISPR/Cas9 Phaeodactylum tricornutum mutant through whole genome resequencing. (2018). https://doi.org/10.7717/peerj.5507

A CRISPR/Cas9 system adapted for gene editing in marine algae. (2016). https://doi.org/10.1038/srep24951

Other examples of use of CRISPR/Cas9 in P. tricornutum:

The Myb-like transcription factor phosphorus starvation response (PtPSR) controls conditional P acquisition and remodelling in marine microalgae. (2020). https://doi.org/10.1111/nph.16248

Loss of ALBINO3b Insertase Results in Truncated Light-Harvesting Antenna in Diatoms. (2019). https://doi.org/10.1104/pp.19.00868

 
  • TALENs

Transcription activator-like effector nucleases (TALENs) have been used to edit P. tricornutum as well. This section will be expanded in the future.

Some reference articles to read if you wish to use TALENs:

A strategy to complement PtAUREO1a in TALEN knockout strains of Phaeodactylum tricornutum. (2019). https://doi.org/10.1016/j.algal.2019.101469

A fast and reliable strategy to generate TALEN-mediated gene knockouts in the diatom Phaeodactylum tricornutum. (2017). https://doi.org/10.1016/j.algal.2017.02.005

PtAUREO1a and PtAUREO1b knockout mutants of the diatom Phaeodactylum tricornutum are blocked in photoacclimation to blue light. (2017). https://doi.org/10.1016/j.jplph.2017.05.020

Inactivation of Phaeodactylum tricornutum urease gene using transcription activator-like effector nuclease-based targeted mutagenesis. (2015). https://doi.org/10.1111/pbi.12254

 
  • RNA Interference

Use of antisense and inverted stem-loop constructs to target genes for knockdown using RNA interference has been a very effective strategy for modifying gene expression in P. tricornutum. This section will be expanded in the future.

Some reference articles to read if you wish to use RNAi:

Reduced vacuolar beta-1,3-glucan synthesis affects carbohydrate metabolism as well as plastid homeostasis and structure in Phaeodactylum tricornutum. (2018). https://doi.org/10.1073/pnas.1719274115

  • Knockdown of PtBGS in using antisense RNAi construct.

A lipid droplet-associated protein involved in lipid droplet biogenesis and triacylglycerol accumulation in the oleaginous microalga Phaeodactylum tricornutum. (2017). https://doi.org/10.1016/j.algal.2017.07.028

  • Knockdown of PtLDP1 in using RNAi.

Identification of a triacylglycerol lipase in the diatom Phaeodactylum tricornutum. (2016). https://doi.org/10.1016/j.bbalip.2015.12.023

  • Knockdown of a novel TAG lipase in using RNAi.

Knockdown of phosphoenolpyruvate carboxykinase increases carbon flux to lipid synthesis in Phaeodactylum tricornutum. (2016). https://doi.org/10.1016/j.algal.2016.02.004

  • Knockdown of PEPCK in using RNAi.

An RNA interference knock-down of nitrate reductase enhances lipid biosynthesis in the diatom Phaeodactylum tricornutum. (2015). https://doi.org/10.1111/tpj.13052

  • Knockdown of nitrate reductase in using RNAi.

 
 
 

Other Resources

  • Plasmid Assembly

  • Resources for Synthetic Chromosomes

This section will be expanded in the future to include methods for plasmid assembly includiing PCR-based yeast assembly, Gateway cloning, Golden Gate assembly, and others.

Chromosome 25 and chromosome 26 from P. tricornutum have been isolated, cloned, assembled, and propogated as episomal vectors in S. cerevisiae. These constructs serve as the start point for the synthetic genome.

Assembly of eukaryotic algal chromosomes in yeast (2013). https://doi.org/10.1186/1754-1611-7-30

Together with the community, the Synthetic Diatoms Project will work toward generating synthetic (and minimal) versions of every chromosome in P. tricornutum. After a compelte resequencing of the P. tricornutum genome to survey the number of chromosomes, working groups will begin to clone and assemble each chromosome.