The present application is filed with sequence listing(s) attached hereto and incorporated by reference.
This invention relates to molecular biology, and more specifically to the transformation of algal cells and the expression of exogenous deoxyribonucleic acid (DNA).
Exemplary embodiments provided herein include novel promoters isolated from the microalgae, Nannochloropsis. These promoters drive gene expression in a bidirectional manner, and are especially useful for the genetic manipulation of Nannochloropsis and other organisms. The inventors herein successfully used these promoters (in both parallel and antiparallel orientations with respect to a Sh ble gene cassette) to impart zeocine-resistance to Nannochloropsis.
Exemplary embodiments provided herein include novel promoters isolated from the microalgae, Nannochloropsis. These promoters drive gene expression in a bidirectional manner, and are especially useful for the genetic manipulation of Nannochloropsis and other organisms. The inventors herein successfully used these promoters (in both parallel and antiparallel orientations with respect to a Sh ble gene cassette) to impart zeocine-resistance to Nannochloropsis.
FIGS. 1A-1B show two exemplary transformation constructs, transformation construct NT6 (
When analyzing a Nannochloropsis genomic sequence, the inventors found two divergently transcribed Vcp genes orientated back to back (i.e. transcription must have been initiated from the nucleotide sequence separating both genes) separated by several hundred nucleotides. The inventors believed that this nucleotide sequence that separated the divergently transcribed Vcp genes included the requisite regulatory elements to drive expression of both divergently transcribed Vcp genes.
The inventors created transformation constructs NT6 and NT7 to confirm they had discovered a bidirectional promoter in the Nannochloropsis genome. The bidirectional promoter was amplified from the Nannochloropsis genome using Polymerase Chain Reaction (PCR) and other standard techniques. A Nannochloropsis transformation construct (or vector) was constructed using a pJet vector as the backbone. The bidirectional promoter was cloned in both a parallel (NT6) and an anti-parallel (NT7) fashion relative to a standard zeocine-resistance (Sh ble) cassette. A Vcp 3′-UTR was placed immediately downstream of the zeocine-resistance (Sh ble) cassette in both constructs.
The NT6 and NT7 transformation constructs were cut out via restriction enzyme digestion and the transformation construct purified after DNA gel electrophoreses. A PL90 transformation construct, as described in U.S. Non-Provisional patent application Ser. No. 12/480,635 filed on Jun. 8, 2009, titled “VCP-Based Vectors for Algal Cell Transformation,” that included another Vcp-promoter was linearized to be used as a comparison to the NT6 and NT7 transformation constructs. All three transformation constructs in equimolar amounts were used to transform Nannochloropsis cells, which were then allowed to incubate at room temperature under ˜85 μE light on solid selective media (with a zeocine concentration of 2 micrograms per milliliter) for several weeks until visible colonies were formed.
Referring again to
Zeocine was utilized in a kill-curve as follows:
1. 1 mL aliquots of F2 media were added to wells in a 24-well plate.
2. Zeocine was added to the wells with F2 media to achieve final concentrations of zeocine as listed in the left-hand column of the
3. Colonies were randomly picked from agar plates containing the NT6, NT7, and PL90 transformants (the number of transformants tested for each transformation construct is given as the final line-item in the
4. Each colony was resuspended in 30 uL of N2 media.
5. 2 uL of each colony-resuspension was added to the wells containing the increasing amounts of zeocine (wells without zeocine were included as controls, but are not shown in the
6. The 24-well plates were allowed to incubate under 85 uE light for 1 week.
7. Optical density measurements (at 750 nm) for each well were obtained with a spectrophotometer.
8. For each transformant, the highest zeocine concentration was determined required to enable at least 50% growth of the cell line as compared to the no-zeocine controls.
The exemplary data reflected in the
The various exemplary bidirectional promoters provided herein have been used to drive expression of genes introduced to Nannochloropsis via transformation. They also may be used to perform activation-tagging random insertional mutagenesis experiments. To achieve certain phenotypes through genetic manipulation, up-regulation of the expression of certain genes (as opposed to the down-regulation or the knocking out of certain genes) may be required. Forward genetics may be performed with the bidirectional promoter in a highly efficiently manner because the promoter can activate genes in both directions. A typical activation tagging experiment, to achieve higher oil production, could be performed as follows: A transformation construct comprising the bidirectional promoter as depicted in
The various exemplary bidirectional promoter sequences provided herein may be used to perform RNA-interference (RNAi). RNAi is based on the presence of dsRNA, either introduced exogenously or produced within a cell itself. The bidirectional promoter provides for a facile system to perform RNAi, as the gene of interest can be expressed in parallel and anti-parallel fashions, thus making reverse complements of one another (dsRNA).
This application claims the benefit and priority of U.S. Provisional Patent Application Ser. No. 61/207,564 filed on Feb. 13, 2009, titled “Bidirectional Promoter in Nannochloropsis,” which is incorporated by reference herein. This application is related to U.S. Non-Provisional patent application Ser. No. 12/480,635 filed on Jun. 8, 2009, titled “VCP-Based Vectors for Algal Cell Transformation,” which in turn claims the benefit and priority of U.S. Provisional Patent Application Ser. No. 61/059,672 filed on Jun. 6, 2008, titled “VCP-Based Vector For Nannochloropsis Transformation,” the disclosures of both which are incorporated by reference herein.
Number | Date | Country | |
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61207564 | Feb 2009 | US |