P. kessleri-I strain was scaled up using liquid f/2 medium. The culture was centrifuged at 3600xg and the pellet was resuspended in 3ml ice cold breaking buffer containing 5mM dithiothreitol (DTT) and 5mM sodium ascorbate. Algal cells were sonicated on ice using a cell disruptor (Sonics, 3 mm Tapered microtip probe) at frequency of 1 KHz, 5 sec/ 5 sec off pulse at 30% amplitude for 2 min. The algal cells were collected by centrifugation and pellet was resuspended in 1 ml buffer containing 10 mM HEPES, 600 Mm sorbitol, 50 mM MgCl2 and 0.1% bovine serum albumin (BSA), pH 7.5–7.8 at 4°C. The autofluorescence of intact chloroplasts was monitored using Fluorescence Brightner 28 (Sigma Aldrich). The fluorescence from stained cellular components was observed at 650 nm and autofluorescence from intact chloroplast was seen at 420 nm. The presence or absence of fluorescence was observed using a NIKON fluorescence microscope. After, confirmation of chloroplast release, the enriched chloroplast was layered on 60–70% sucrose gradient. The gradients were allowed to equilibrate overnight at 4°C. Gradient layered with cell lysate was centrifuged at 197120xg for 60 min at 10°C using the Ultracentrifuge (Beckman Coulter Optima XE–100). The chloroplast fraction was observed as a single dark green band almost at the middle of the tube (41). The plastomic DNA was extracted using CTAB (Cetyl Trimethyl Ammonium Bromide) buffer method (42). The quality of genomic DNA was checked on 1% agarose gel for a single intact band. 1µl of sample was used for determining the DNA concentration by NanoDrop™ 2000 Spectrophotometer (Thermo Scientific™). The purified cpDNA was supplied to Xcelris Genomics (Ahmedabad, India) for sequencing the chloroplast genome of P. kessleri-I.
The plastome of P. kessleri-IMN013385) and Parachlorella kessleri (NC_012978.1) was compared using MAUVE software: Multiple alignment of conserved genomic sequence with rearrangements (30). Progressive MAUVE genome alignment algorithm was used with default parameters in order to compare the two genomes so as to observe any gene loss, duplication, genome rearrangement, and horizontal transfer caused due to recombination.
5.6. Study of antibiotics sensitivity
Prior to chloroplast transformation, algal cells were adapted to grow on TAP medium (47) without addition of NaCl (salt) so that sensitivity to antibiotics can be accurately determined. In the presence of salt, the algal cells may become tolerant to antibiotics (36). In order to identify an optimal concentration of the antibiotics zeocin (InvitroGen) and spectinomycin (Sigma Aldrich), different concentrations of zeocin (10, 20, 40, 60, 80 and 100 mg/L) and spectinomycin (100, 200, 300, 400, 500 and 600 mg/L) were tested using TAP medium solidified with 1.2% agar. Approximately 1.5 × 106cells of P. kessleri-I were plated onto 90 mm petri plates and incubated with 16:8 h light (irradiance of 2000 lux) and dark conditions at 25±1°C. The final concentrations of 20 mgL–1 zeocin and 400 mgL–1 spectinomycin were observed to completely cease the growth of P. kessleri-I cells. Therefore, 20 mgL–1 zeocin and 400 mgL–1 spectinomycin were taken into account as optimized antibiotic concentrations for exploring the chloroplast transformation in P. kessleri-I.
5.7. Chloroplast transformation vector
The chloroplast genome sequences were used for generating the flanking regions in-silico. Both left and right flanks contained the plastome fragments approx. 0.8 Kb of each, comprising of 16S rDNA and trnI and trnA and 23S rDNA respectively. Between the right and left flanks, transgenes cassette PkCpV contained 16S Prrn promoter-g10-ShBle gene–3’UTR-Trps::Promoter psbA–5’UTR-psbA-aadA gene–3’UTR-psbA was cloned into the pUC19 plasmid as shown in Fig. 3A. The whole DNA cassette of PkCpV vector prepared in-silico and DNA vector was synthesised with the help of vendor (BioBasic Inc., Canada). The PkCpV chloroplast transformation vector was transformed into E. Coli (Top–10) competent cells to maintain and bulk up the plasmid.
5.8. Genetic transformation of P. kessleri-I and antibiotics selection
Prior to bombardment, algal cells lawn was prepared, containing high concentration of cells in the middle area of the Petri-plates. The chloroplast transformation vector DNA specific to P. kessleri-I (PkCpV) was coated on gold carrier particles S550d following vendor’s instruction (Seashell Technology, CA, USA). About 10 μg of plasmid DNA (stock 1mg/ml) was added to 3 mg of gold carrier for 5 transfections. Thereafter, an equal volume of precipitation buffer (50 μL) was added. The total mixture (100 μL) was briefly vortex and incubated for 3 minutes. The mixture was spun (10,000 rpm for 10 sec) to pellet the DNA coated on gold particles. The supernatant was removed and 500 μL of 100% chilled ethanol was added. Again mixture was briefly vortex and spun (10,000 rpm for 10 sec) to pellet DNA-coated gold particles. Supernatant was discarded and 50 μL of 100% ethanol was added to DNA-coated gold particles. The pellet was homogenised in ethanol by sonication and 10 μL each of the suspension of DNA-coated gold particles was spotted in the middle of microcarrier (10 discs) (Bio-Rad, Hercules, CA, USA). The DNA gold particles were bombarded on the algal cells lawn using 1350 psi rupture disc at the 6 and 9 cm distance respectively (31). After overnight incubation of cultures in dark, bombarded cells were divided equally on TAP medium containing 20 mgL–1 zeocin and 400 mgL–1 spectinomycin.
5.9. PCR and Southern blot analysis
Transgenic colonies appeared only spectinomycin (400 mg/L) selection medium. No transgenic colonies were observed on TAP medium containing 20 mgL–1 zeocin. Spectinomycin resistant colonies were screened by colony PCR using gene specific primer pair F1 (5’-GTCGACATGGAACAGAAGTTGATT–3’) and R1 (5’-GTGGACAAATTCTTCCAACTGATCT–3’). Each PCR reaction was performed in 50μl reaction mixture containing 1X Taq Reaction buffer (HiMedia Labs, Mumbai, India), 0.1μg DNA template, 200 μM of each dNTPs, 1 Unit Taq polymerase (HiMedia Labs, Mumbai, India), and concentrations of F1 and R1 used 0.5 μM of each oligo. A BioRad MyCycler (BioRad, USA) was employed for PCR amplification as shown in Fig. 3C to amplify about 850 bp amplicon. A PkCpV plasmid DNA was used as a control (25-ng) along with wild type algal cells and transformed colonies selected on 400 mg/L spectinomycin. The amplified PCR products were obtained using 35 cycles at 95°C denaturation of DNA template, 59°C annealing and extension at 72°C for 1 min and final extension at 72°C for 10 min. PCR products were analyzed on agarose gel (1.0 %) electrophoresis (BioRad, USA).
The transgenes integration was further confirmed by Southern blot analysis. Genomic DNA of transformed and untransformed cells was extracted using the DNeasy plant mini kit (Qiagen). About 6 μg genomic DNA was digested with NcoI and HindIII (restriction enzyme located outside the flanking region of 23S) as shown in Fig. 3A. The digested genomic DNA was electrophoresed on 0.8 % agarose gel, denatured and blotted onto Hybond positive nylon membrane. Probe was prepared as per the AlkPhos Direct Labeling Reagents kit (GE Healthcare) using aadA gene around 400 bp fragment isolated from PkCpV plasmid after digestion with NotI and PvuI enzymes. Southern hybridization was carried out according to AlkPhos kit instructions (GE Healthcare, Little Chalfont, United Kingdom).