Genetic Tools

Plasmids and Parts

  • Episomal Plasmids

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

 

  • Including the yeast CEN6-ARSH4-HIS3 sequence

  • Including a low GC content region similar to T. pseudonana centromeres

One way for episomal plasmids to be delivered into T. pseudonana 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, NAT selection marker (nourseothricin 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

  • Integrative Plasmids

Transformed plasmids that integrated randomly into the diatom genome, using a microparticle bombardment or biolistic method. 

Drives constitutive expression using the FCP promoter and cloning site with EcoRV, SphI, and NotI sites for easy insertion of the desired transgene.

Molecular genetic manipulation of the diatom Thalassiosira pseudonana (Bacillariophyceae). 2006. https://doi.org/10.1111/j.1529-8817.2006.00269.x

Drives inducible expression using the nitrate reductase promoter (inducible with nitrate added to media) and cloning site with EcoRV, SphI, and NotI sites for easy insertion of the desired transgene.

Molecular genetic manipulation of the diatom Thalassiosira pseudonana (Bacillariophyceae). 2006. https://doi.org/10.1111/j.1529-8817.2006.00269.x

  • Promoters

Promoters used for T. pseudonana:

Molecular genetic manipulation of the diatom Thalassiosira pseudonana (Bacillariophyceae). 2006. https://doi.org/10.1111/j.1529-8817.2006.00269.x

  • Nitrate Reductase - Nitrogen Inducible (Sequence)

Nitrate as the sole nitrogen source in the medium, and is inactivated in the presence of ammonium ions.

Molecular genetic manipulation of the diatom Thalassiosira pseudonana (Bacillariophyceae). 2006. https://doi.org/10.1111/j.1529-8817.2006.00269.x

Downstream gene is upregulated in silicon deprived conditions.

Development of a silicon limitation inducible expression system for recombinant protein production in the centric diatoms Thalassiosira pseudonana and Cyclotella cryptica. (2017). https://doi.org/10.1186/s12934-017-0760-3

  • Selection Markers

  • sh ble - Zeocin Resistance 

  • nat - Nourseothricin Resistance

  • GFP - Green Fluorescent Protein 

Molecular genetic manipulation of the diatom Thalassiosira pseudonana (Bacillariophyceae). 2006. https://doi.org/10.1111/j.1529-8817.2006.00269.x

 
 

Editing and RNAi

 
 
 
 
  • CRISPR/Cas9

Golden Gate Level 0 cassette encoding the U6 promoter.

Editing of the urease gene by CRISPR-Cas in the diatom Thalassiosira pseudonana. (2016). https://doi.org/10.1186/s13007-016-0148-0

Golden Gate Level 1 cassette encoding a Nat selectable marker under the FCP promoter.

Editing of the urease gene by CRISPR-Cas in the diatom Thalassiosira pseudonana. (2016). https://doi.org/10.1186/s13007-016-0148-0

Golden Gate Level 1 cassette encoding a Cas9-YFP fusion under the FCP promoter.

Editing of the urease gene by CRISPR-Cas in the diatom Thalassiosira pseudonana. (2016). https://doi.org/10.1186/s13007-016-0148-0

Golden Gate Level 2 construct encoding Cas9-YFP and 2 urease-targeting gRNAs.

Editing of the urease gene by CRISPR-Cas in the diatom Thalassiosira pseudonana. (2016). https://doi.org/10.1186/s13007-016-0148-0

  • RNAi

RNA interference is a useful tool for temporal or tissue-specific down regulation of certain genes at the level of translation. An integrative plasmid with an antisense gene will bind the native sense strand, creating a dsRNA product targeted for silencing. 

Constructs using the integrative plasmids from:

Molecular genetic manipulation of the diatom Thalassiosira pseudonana (Bacillariophyceae). 2006. https://doi.org/10.1111/j.1529-8817.2006.00269.x

 

At the NotI insertion sites in each plasmid, the silicon antisense gene was inserted. 

A role for the cell-wall protein silacidin in cell size of the diatom Thalassiosira pseudonana. (2017). https://doi.org/10.1038/ismej.2017.100

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Programmable genome editing is a valuable tool with applications in gene knockouts and modifications. These plasmid constructs utilize Golden Gate cloning with specific restriction sites for ease of modifying the sgRNA in the plasmid. These sites are also made for the promoters, Cas9, and NLS sites. Editing at these various levels in cloning (shown left) allow flexibility of the final CRISPR/Cas9 editing cassette. 

This method was presented by Hopes et al. in 

Editing of the urease gene by CRISPR-Cas in the diatom Thalassiosira pseudonana. (2016). https://doi.org/10.1186/s13007-016-0148-0