Strains and culture conditions
The wild-type P. morum strain SAG 32.96 was obtained from the Culture Collection of Algae at Goettingen University (SAG), Germany. Cultures were maintained in Jaworski medium (JM) [47] at 29°C in an 8 h dark/16 h light (~10,000 lux) cycle [11, 20]. Cultures were grown in 10 ml glass tubes, 50 or 300 ml Erlenmeyer flasks, or 1,000 ml Fernbach flasks. The glass tubes had caps that allow for gas exchange, and Erlenmeyer and Fernbach flasks were aerated via Pasteur pipettes with approximately 50 cm3 sterile air/min [11, 20]. Transgenic strains that express the aphVIII gene were grown in JM in the presence of 1 μg paromomycin/ml (paromomycin sulfate, Sigma-Aldrich, St. Louis, MO).
Transformation vectors
The pPmr3 plasmid (Figure 3A) contains the 0.8 kb coding region of the S. rimosus aphVIII selectable marker gene, a V. carteri hsp70A-rbcS3 tandem promoter (0.5 kb of V. carteri hsp70A and 0.27 kb of V. carteri rbcS3 sequences), the 3′ UTR from the V. carteri rbcS3 gene (0.53 kb of downstream sequence), and the pBluescript II vector backbone [GenBank: AY429514] [31]. The total size of the pPmr3 plasmid is 5.1 kb.
The pPsaD-GLuc plasmid (Figure 3B) contains the 0.57 kb coding region of the luciferase (gluc) gene from G. princeps, which was engineered to match the codon usage in C. reinhardtii, 0.8 kb of upstream sequence, which includes the C. reinhardtii PSAD promoter, the 3′-UTR of the C. reinhardtii psaD gene (0.56 kb of downstream sequence), and the pBluescript vector backbone [27, 36] [GenBank: EU372000, AF335592]. The pPsaD-GLuc plasmid is 5.0 kb in size.
The pHsp70A-GLuc plasmid (Figure 3C) contains the 0.57 kb coding region of the luciferase (gluc) gene from G. princeps (codon-optimized for C. reinhardtii) [27] fused downstream of a 0.8 kb genomic DNA fragment that contains the first three exons and two introns of the HSP70B gene of C. reinhardtii; this fragment of the HSP70B gene contains the sequence coding for the chloroplast transit peptide of HSP70B. The hybrid HSP70B/gluc gene is driven by the C. reinhardtii HSP70A promoter (0.26 kb of upstream sequence) and the 3′ UTR comes from the C. reinhardtii RBCS2 gene (0.22 kb of downstream sequence) [27]. The total size of the pHsp70A-GLuc plasmid is 4.9 kb.
The pHRLucP plasmid (Figure 3D) contains the 0.57 kb coding region of the luciferase (gluc) gene of G. princeps, which was engineered to match the codon usage in C. reinhardtii, the V. carteri hsp70A-rbcS3 tandem promoter (0.5 kb of V. carteri hsp70A and 0.27 kb of V. carteri rbcS3 sequences), the C. reinhardtii PSAD 3′ UTR (0.55 kb of downstream sequence), and the pBluescript II vector backbone [11, 27, 36]. The pHRLucP plasmid is 5.0 kb in size.
Preparation of plasmid DNA
Plasmid DNA was purified using the Nucleospin® Plasmid Kit according to the manufacturers’ instructions (Macherey-Nagel, Düren, Germany).
Coating of microprojectiles
Gold microprojectiles (0.6 μm in diameter, Bio-Rad, Hercules, CA) were coated with the required plasmids for biolistic transformation as previously described [11, 20]. The DNA-coated microprojectiles were resuspended in 60 μl of ethanol and kept at 4°C; these microprojectiles were used within 3 h of preparation.
Determination of cell concentration
In P. morum the number of cells per colony varies. Therefore, we refer to “cells/ml” rather than “colonies/ml”. The concentration of cells was determined using a hemacytometer with Neubauer ruling.
Stable nuclear transformation by particle bombardment
The stable transformation of P. morum was performed using a Biolistic® PDS-1000/He (Bio-Rad) particle gun. To this end, a logarithmically growing P. morum culture (150 ml) at a cell concentration of ~7 × 104 cells/ml was harvested by centrifugation (1,000 g, 5 min, swing-out rotor) and resuspended in 6 ml of JM. One milliliter of the suspension was evenly spread on a cellulose acetate membrane filter and excess liquid was removed as previously described [11, 20]. One-sixth of the DNA-coated microprojectiles were evenly spread on a macrocarrier (Bio-Rad) that was placed in a macrocarrier holder (Bio-Rad). The transformation procedure was as previously described [11, 20] and the most successful combination of parameters is shown in Table 1. After the bombardment step, the algae were washed off from the membrane filter with JM. The procedure was repeated five times and the algae from six rounds of bombardment were pooled and then evenly distributed among twelve 50 ml Erlenmeyer flasks containing a volume of ~40 ml of JM. The bombarded colonies were incubated under standard conditions for 24 h to allow for regeneration and expression of the transgenes. Then, 2 μg of paromomycin/ml was added. The large fraction of non-transformed cells died within 48 h and the medium clarified. After 10–14 days of incubation in the presence of antibiotic, re-greening of flasks indicated the initial presence and reproduction of at least one paromomycin-resistant P. morum transformant that led to a population of transformants. No more than one transformant per flask was analyzed [11, 20].
Re-isolation of transformants
Transformants were re-isolated to ensure uniform genetic conditions for all further analyses. For this purpose, a serial dilution of an exponentially growing P. morum culture was performed in a Terasaki plate (Nunc™ MicroWell™ MiniTrays; Thermo Fisher Scientific, Langenselbold, Germany) [11, 20]. Each well of the Terasaki plate was filled with 10 μl of JM. A single P. morum colony was finally transferred into a standard glass tube with JM, containing 1 μg of paromomycin/ml. Further incubation was under standard conditions.
Paromomycin-resistance assay
Cells of transformed or wild-type P. morum strains were transferred into the wells of a 24-well culture plate (Sarstedt, Nümbrecht, Germany) with a wide range of paromomycin concentrations from 0 to 100 μg/ml in 1 ml JM (concentrations are given in Figure 2). At the beginning of the assay, each well contained approximately 4,000 cells, which corresponds to 250–300 colonies. After incubation under standard conditions for 12 days, the wells were analyzed for viable green cells or white remains of lysed cells.
Primer design
Oligonucleotide primers were designed using the primer analysis software Oligo 6 (Molecular Biology Insights, Cascade, CO), DNASIS™ (version 7.00; Hitachi Software Engineering, San Francisco, CA), and Primer Express® (Applied Biosystems, Foster City, CA).
Isolation of genomic DNA
Ten milliliters of a logarithmically growing P. morum culture with a density of 6 × 106 cells/ml was harvested by centrifugation (3,500 g for 10 min). The resulting pellet, which had a wet weight of ~80 mg, was washed with water and resuspended in 40 μl water. Isolation of genomic DNA was as previously described [11, 20].
Genomic PCR
PCR reactions with genomic DNA as a template were carried out in a total volume of 50 μl that contained ~100 ng of genomic DNA, 300 nM of each primer, 0.2 mM dNTP, 1.5 mM MgCl2, and 2.6 units of Expand High Fidelity enzyme mix in 1× Expand High Fidelity buffer (Roche Applied Science, Basel, Switzerland). The PCR reactions were performed on a T3 Thermocycler PCR system (Biometra, Göttingen, Germany) using the following conditions: 40 cycles of 94°C for 20 s, 55°C for 30 s, and 72°C for 45 s and a final extension was at 72°C for 10 min. The PCR products were cloned and sequenced (Eurofins, Ebersberg, Germany).
Isolation of total RNA
Total RNA was extracted from ~100 mg of concentrated, frozen P. morum algae using 1 ml of phenol-based TRI Reagent (Sigma-Aldrich, St. Louis, MO) and 300 μl trichloromethane. RNA precipitation and RNA purification was as previously described [11, 20].
Reverse Transcription (RT)-PCR
First strand cDNA synthesis was carried out using 1 μg of total RNA and Moloney murine leukemia virus (MMLV) reverse transcriptase lacking ribonuclease H activity (H minus) according to the manufacturer’s instructions (Promega). The subsequent PCR step was performed using the Expand High Fidelity PCR system (Roche Applied Science, Basel, Switzerland), a T3 Thermocycler PCR system (Biometra), and the following cycling conditions: 40 cycles of 94°C for 20 s, 55°C for 30 s, and 72°C for 45 s and a final extension at 72°C for 10 min. The RT-PCR products were cloned and sequenced.
Induction of luciferase activity
Preliminary tests showed that the shift of a P. morum culture (3–6 × 105 cells/ml) from 29°C to 39°C for 1 h, followed by a recovery phase at 29°C for 15 min, leads to the strongest induction of luciferase activity in the temperature range investigated (27°C to 51°C).
Luciferase assays
For assays on light-sensitive films, P. morum cultures with a cell density of 3–6 × 105 cells/ml were divided into aliquots of 50 ml. One aliquot was incubated at the optimal temperature for heat stress-induced expression of luciferase (39°C) for 1 h, and another aliquot was incubated at 29°C as a reference control. Further treatment and execution of the luciferase enzyme assay was as previously described [11, 20]. The final exposure to a chemiluminescence sensitive film (Retina XBA; Fotochemische Werke) was for 2 h at 20°C [11, 20, 35].
The quantification of luciferase activity was conducted as previously described [20, 27] using a MiniLumat LB9506 luminometer (Berthold, Bad Wildbad, Germany). For this purpose, aliquots of a P. morum culture (3–6 × 105 cells/ml) were heat-stressed at 39°C or kept at 29°C (control), respectively. Further treatment was as previously described [20]. Luminescence was recorded as relative light units (rlu). Induction factors were calculated by comparison of samples from heat-shocked versus non-heat-shocked cultures.
In-gel activity assay
One liter of logarithmically growing, heat-stressed or untreated cultures of transformed or wild-type P. morum cultures with cell densities of 4–7 × 105 cells/ml were harvested by filtration on a 10-μm mesh nylon screen. Cells passing the screen were harvested by centrifugation (4,000 g, 5 min) and added to cells collected on the screen. Cells lysates were produced as previously described [11]. The protein concentration of the cell extracts was determined photometrically using the Bio-Rad Protein Assay Dye Reagent (Bio-Rad). For gel electrophoresis, 50 μg of total protein was loaded onto a standard SDS-polyacrylamide gel. The gel electrophoresis, the in-gel renaturation, the execution of the luciferase enzyme assay and the light detection on a chemiluminescence sensitive film was performed as previously described [11].