US20130131330A1 - Bidirectional promoters in nannochloropsis - Google Patents

Bidirectional promoters in nannochloropsis Download PDF

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US20130131330A1
US20130131330A1 US13/681,408 US201213681408A US2013131330A1 US 20130131330 A1 US20130131330 A1 US 20130131330A1 US 201213681408 A US201213681408 A US 201213681408A US 2013131330 A1 US2013131330 A1 US 2013131330A1
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nannochloropsis
bidirectional promoter
bidirectional
transformation
zeocine
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Oliver Kilian
Bertrand Vick
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts

Definitions

  • 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.
  • FIG. 1A shows exemplary transformation construct NT6.
  • FIG. 1B shows exemplary transformation construct NT7.
  • FIG. 2 shows a table reflecting the exemplary results of a growth assay used to analyze the transformants that resulted from the three transformation constructs, NT6, NT7, and PL90.
  • 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 ( FIG. 1A ) and transformation construct NT7 ( FIG. 1B ).
  • Transformation construct NT6 as shown in FIG. 1A includes a bidirectional promoter sequence 110 (SEQ. ID. NO. 1), a Sh ble gene cassette sequence 120 , and a 3′ untranslated region sequence (UTR) 130 .
  • Transformation construct NT7 as shown in FIG. 1B includes a bidirectional promoter sequence 110 (SEQ. ID. NO. 2), a Sh ble gene cassette sequence 120 , and a 3′ untranslated region sequence (UTR) 130 .
  • the inventors 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 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.
  • 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.
  • FIG. 2 shows a table reflecting the exemplary results of a growth assay used to analyze the transformants that resulted from the three transformation constructs, NT6 ( FIG. 1A ), NT7 ( FIG. 1B ), and PL90.
  • the inventors found that the bidirectional promoter sequence 110 ( FIGS. 1A-1B ) in both orientations (i.e. parallel and anti-parallel) within each transformation construct (i.e. NT6 and NT7) drove much higher levels of gene expression than the Vcp-promoter used in the PL90 transformation construct.
  • the growth assay utilized zeocine, an antibiotic that kills most aerobic cells by binding and cleaving the DNA in the aerobic cells.
  • the Sh ble gene product produced by the Sh ble gene cassette sequence 120 ( FIGS. 1A-1B ) prevents toxicity by binding to zeocine and inactivating it. Accordingly, a higher level of Sh ble-transgene expression in an algal cell will lead to a greater ability for the algal cell to survive in higher zeocine concentrations.
  • Zeocine was utilized in a kill-curve as follows:
  • Zeocine was added to the wells with F2 media to achieve final concentrations of zeocine as listed in the left-hand column of the FIG. 2 table (i.e. the final concentrations of zeocine ranged from 0 ug/ml to 200 ug/ml).
  • the 24-well plates were allowed to incubate under 85 uE light for 1 week.
  • 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 FIG. 2 table shows the number of colonies that had at least 50% survival up to the specified zeocine concentration.
  • the data in the FIG. 2 table shows that the NT6 and NT7 transformation constructs withstood higher levels of zeocine than did the PL90 transformation construct. Because the promoter was the only variable between the NT6/NT7 and PL90 transformation constructs, it followed that the increased survival rate of the NT6/NT7 transformation constructs was due to the bidirectional promoter, which is apparently stronger than the Vcp-promoter used in the PL90 transformation construct. It should be noted that wild-type Nannochloropsis will generally not survive zeocine concentrations of 2 micrograms per milliliter and higher.
  • 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 FIG. 1A or 1 B, a selection gene (e.g.
  • the Sh ble gene is isolated via PCR or restriction digest of the plasmid containing the bidirectional promoter would be introduced into Nannochloropsis via previously-described methods.
  • the bidirectional promoter would each be inserted into the Nannochloropsis genome in random locations when individual transformants are analyzed, and at some frequency, it would insert upstream of the genes involved in, e.g., lipid biosynthesis.
  • the Vcp promoters being strong promoters, would likely drive higher expression of the gene(s).
  • An assay such as the Nile Red assay, could be performed to identify transformants that produce more lipids.
  • RNAi RNA-interference
  • 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).

Abstract

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.

Description

    CROSS-REFERENCE TO RELATED APPLICATIONS
  • This continuation application claims the benefit and priority of U.S. Non-Provisional patent application Ser. No. 12/706,683 filed Feb. 16, 2010, titled “Bidirectional Promoter in Nannochloropsis,” which 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,” all of which are incorporated by reference herein.
  • This continuation 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.
    • REFERENCE TO SEQUENCE LISTINGS
  • The present application is filed with sequence listing(s) attached hereto and incorporated by reference.
    • FIELD OF THE INVENTION
  • This invention relates to molecular biology, and more specifically to the transformation of algal cells and the expression of exogenous deoxyribonucleic acid (DNA).
    • SUMMARY OF THE INVENTION
  • 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.
    • BRIEF DESCRIPTION OF THE DRAWINGS
  • FIG. 1A shows exemplary transformation construct NT6.
  • FIG. 1B shows exemplary transformation construct NT7.
  • FIG. 2 shows a table reflecting the exemplary results of a growth assay used to analyze the transformants that resulted from the three transformation constructs, NT6, NT7, and PL90.
    •  DETAILED DESCRIPTION OF THE INVENTION
  • 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 (FIG. 1A) and transformation construct NT7 (FIG. 1B). Transformation construct NT6 as shown in FIG. 1A includes a bidirectional promoter sequence 110 (SEQ. ID. NO. 1), a Sh ble gene cassette sequence 120, and a 3′ untranslated region sequence (UTR) 130. Transformation construct NT7 as shown in FIG. 1B includes a bidirectional promoter sequence 110 (SEQ. ID. NO. 2), a Sh ble gene cassette sequence 120, and a 3′ untranslated region sequence (UTR) 130.
  • 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.
  • FIG. 2 shows a table reflecting the exemplary results of a growth assay used to analyze the transformants that resulted from the three transformation constructs, NT6 (FIG. 1A), NT7 (FIG. 1B), and PL90. The inventors found that the bidirectional promoter sequence 110 (FIGS. 1A-1B) in both orientations (i.e. parallel and anti-parallel) within each transformation construct (i.e. NT6 and NT7) drove much higher levels of gene expression than the Vcp-promoter used in the PL90 transformation construct.
  • Referring again to FIG. 2, the growth assay utilized zeocine, an antibiotic that kills most aerobic cells by binding and cleaving the DNA in the aerobic cells. The Sh ble gene product produced by the Sh ble gene cassette sequence 120 (FIGS. 1A-1B) prevents toxicity by binding to zeocine and inactivating it. Accordingly, a higher level of Sh ble-transgene expression in an algal cell will lead to a greater ability for the algal cell to survive in higher zeocine concentrations.
  • 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 FIG. 2 table (i.e. the final concentrations of zeocine ranged from 0 ug/ml to 200 ug/ml).
  • 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 FIG. 2 table).
  • 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 FIG. 2 table).
  • 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 FIG. 2 table shows the number of colonies that had at least 50% survival up to the specified zeocine concentration. The data in the FIG. 2 table shows that the NT6 and NT7 transformation constructs withstood higher levels of zeocine than did the PL90 transformation construct. Because the promoter was the only variable between the NT6/NT7 and PL90 transformation constructs, it followed that the increased survival rate of the NT6/NT7 transformation constructs was due to the bidirectional promoter, which is apparently stronger than the Vcp-promoter used in the PL90 transformation construct. It should be noted that wild-type Nannochloropsis will generally not survive zeocine concentrations of 2 micrograms per milliliter and higher.
  • 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 FIG. 1A or 1B, a selection gene (e.g. the Sh ble gene), and an 5′ UTR, is isolated via PCR or restriction digest of the plasmid containing the bidirectional promoter would be introduced into Nannochloropsis via previously-described methods. The bidirectional promoter would each be inserted into the Nannochloropsis genome in random locations when individual transformants are analyzed, and at some frequency, it would insert upstream of the genes involved in, e.g., lipid biosynthesis. Compared to the promoters of genes involved with lipid biosynthesis, the Vcp promoters, being strong promoters, would likely drive higher expression of the gene(s). An assay, such as the Nile Red assay, could be performed to identify transformants that produce more lipids.
  • 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).

Claims (7)

What is claimed is:
1. A bidirectional promoter for a transformation construct for algal cell transformation, the bidirectional promoter comprising the nucleotide sequence of SEQ. ID. NO. 2.
2. The bidirectional promoter of claim 1, wherein the algal cell is of algal genus Nannochloropsis.
3. The bidirectional promoter of claim 1, wherein the bidirectional promoter promotes transcription of a first nucleotide sequence adjacent to a first side of the bidirectional promoter in a 3′ direction and the bidirectional promotes transcription of a second nucleotide sequence adjacent to a second side of the bidirectional promoter in a 5′ direction.
4. The bidirectional promoter of claim 3, wherein the bidirectional promoter promotes transcription of the first and the second nucleotide sequences at a same time.
5. The bidirectional promoter of claim 3, wherein the first nucleotide sequence includes a selection marker gene.
6. The bidirectional promoter of claim 3, wherein the second nucleotide sequence includes a selection marker gene.
7. The bidirectional promoter of claim 5 or claim 6, wherein the selection marker gene is a Sh Ble gene.
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