A rapid and low-cost method for genomic DNA extraction from the cyanobacterium Synechocystis

Abstract A two-step method is reported for preparation of genomic DNA from the model cyanobacterium Synechocystis that can be performed with minimal equipment and reagents in about an hour. High yields of genetic material can be obtained (200–450 ng/μl) with reasonable purity. A further ethanol precipitation step can be included but is not necessary if template is simply required for polymerase chain reaction (PCR) or digestion. This new protocol is helpful for amplification of genes of interest in early-stage research projects and for low throughput screening of transformants. It is more reliable than colony PCR of Synechocystis cultures, and less involved and cheaper than existing clean-DNA preparation methods. It represents an unusually simple and reliable extraction protocol for the growing body of research making use of this cyanobacterium.


Introduction
The increasing interest in cyanobacteria for biotechnology follows their long history as models for the chloroplast [1]. Synechocystis sp. PCC 6803 became a pre-eminent model organism in photosynthesis research as it was the first photoautotrophic organism to have its complete genome sequence published [2] and, in addition, it is naturally transformable [3]. This, and early crystal structures of photosystems from the closely related cyanobacterium Thermosynechococcus elongatus, were key to better understanding of photosynthesis and its regulation [4,5]. Following the early arrival of Synechocystis in molecular biology and photosynthetic research, it maintains its position as an experimentally amenable photoautotroph in the laboratory by virtue of its increasing use in systems biology and biotechnology. An unusually large research base of genomic, biochemical, and physiological data mean that cyanobacteria are considered to provide an excellent genetic framework for synthetic biology [6,7] and for drug development [8][9][10] by virtue of their native anticancer and proapoptotic compounds, along with their overpro-duction of phenylpropanoid precursors [11,12]. Their use in sustainable bioenergy research has been an area of particular activity [13][14][15] including production of bioethanol [16] or hydrogen [17], and they have been explored as workhorses for bioplastic production (for review, see Katayama et al. [18]).
Because hundreds of studies using this model organism have been published annually for decades, it is also possible to evaluate and compare data from different laboratories and strains for informed planning and scale-up. Meanwhile, methods for use with Synechocystis have been optimized for many years. DNA extraction remains a practical challenge for many people engaged in cyanobacterial research, however. Sufficient yield and quality are required for repeated use of genomic DNA as template in PCR, in order to feed amplicons through mutations, insertions, or deletions in cloning vectors. A rapid and efficient mechanism is also required for the analysis of DNA from transformants. It is noticeable in performing rapid DNA extraction from transformed Arabidopsis thaliana compared with transformed Synechocystis that the former has more reliable "quick and dirty" methods [19,20]. Rapid and reliable extraction of genetic material, ideally with low time and financial commitment and limited chemical hazards, would be of benefit to many Synechocystis projects. Existing cyanobacterial DNA extraction procedures, however, tend to use harmful solvents, labile enzyme stocks, and time-consuming protocols.
The need to break the resistant Synechocystis cell adds an extra step to kit-based methods. The multilayered cell wall and S layer [21] is disrupted in existing procedures by enzymatic lysis [e.g., lysozyme [22], multicomponent buffers [23], or physical means (e.g., glass beads [24])]. The procedure outlined below therefore minimizes the number of steps for the process and avoids costly reagents and multicomponent buffers, by reducing glass bead breaking steps, then adapting one of the simplest methods used for DNA extraction from plants (the "Shorty" prep [20]). PCR and restriction digests on the extracts tested showed it would be possible to use this straightforward protocol to increase efficiency within many Synechocystis research projects.

Rapid DNA extraction
The 40 ml of overnight and long-term Synechocystis cultures (of $2 Â 10 8 cells/ml) was pelleted in 50 ml sterile centrifuge tubes (Fisher, Hampton, USA) at 4000 g for 5 min. The supernatant was removed from each tube and the pellet was resuspended in sterile deionized water, and centrifugation repeated to remove residual medium. The tube containing the washed cell pellet was placed on ice and resuspended in 5 ml extraction buffer [200 mM Tris-HCl pH8.0 (Sigma, Darmstadt, Germany); 0.4 M lithium chloride (Fisher); 25 mM EDTA pH 8 (Sigma); 1% w/v SDS (Applichem, Ottoweg, Germany); pH 9.0]. Approximately 200 ll of sterile acid-washed glass beads (150-212 mm; Sigma) was added to the resuspended pellet, the tube was vortexed for 30 s and then returned to ice for 30 s. This step was repeated 5 times. After centrifuging at 3000 g for 15 min at 4 C, the supernatant was gently taken up into a sterile 10 ml syringe (Beckton Dickinson, Franklin Lakes, USA) and filtered through a sterile 0.2 lm filter (Minisart; Sartorius, Gö ttingen, Germany). From 5 ml pellet in buffer, $4 ml of filtrate was collected in a 5 ml tube. This was split into five aliquots of 800 ll in 1.5 ml microcentrifuge tubes for alcohol precipitation of DNA, when required. This was achieved by adding 600 ll of ice-cold isopropanol (Fisher) and immediate mixing by pipetting. Samples were then centrifuged at 16 000 g for 20 min at 4 C, and the supernatant removed carefully so as not to disturb the pellet. Tubes were left to air dry for 15 min then 200 ll of TE buffer (2 mM Tris-HCl pH 8.0; 1 mM EDTA pH 8.0) was added for resuspension of the pellet. Resuspended material was transferred from each tube to the next in turn, to resuspend each pellet sequentially, and all DNA were collected in one 200 ll aliquot.

Optional purification step
The 10 ll of sterile 3 M sodium acetate (pH 5.2; Sigma) was added to 100 ll of genomic DNA extract and vortexed to mix. The 300 ll of ice-cold absolute ethanol (Fisher) was added before vortexing again and incubating the tube at À20 C for 2 h. Samples were centrifuged at 16 000 g for 30 min at 4 C, and the supernatant was removed. The pellet was washed by adding 200 ll of room temperature 70% ethanol, centrifuging at 10 000 g for min at 4 C, and removing the supernatant. The pellet was again left to air dry for 10 min, before resuspending it in 50 ll of TE pH 8.0. Samples, once resuspended, were centrifuged for 3 min at 5000 g and the supernatant was carefully transferred to a fresh, sterile 1.5 ml tube.

DNA analysis
Purity of DNA was assessed using A 260 /A 230 and A 260 /A 280 values (NanoDrop 2000C spectrophotometer; ThermoFisher, Waltham, MA, USA; Supplementary Fig. S1). DNA quality and quantity were also checked by gel electrophoresis and compared with a bacteriophage lambda digest.

PCR and enzyme digestion
Whether DNA quality was appropriate for use as a template in PCR was assessed in reactions for a standard housekeeping gene (130 bp of the 16 s rRNA gene; 5 0 -AGCGTCCGTAG GTGGTTATG-3 0 and 5 0 -CTACGCATTTCACCGCTACA-3 0 ), and two further test open reading frames with cloning primers containing mismatches for enzyme cut sites (1024 bp product from 5 0 -GCCggattcAGGCCCGTGAATTTCTTAAA-3 0 and 5 0 -CAAggtaccG ATATAGTCCGATAATTTGCT-3 0 ; 620 bp product from 5 0 -CTAgaat tcATTTTTGCTGTAGTAATGC-3 0 and 5 0 AAAGTCAcggccg GCCCCTTCT-3 0 ). PCR was carried out using 1 ll of the extracted DNA (with or without purification), DreamTaq polymerase (ThermoFisher) and RNase/DNase free water (HyPure; GE Life Sciences, Marlborough, MA, USA) in a 25 ll total volume in 0.2 ml PCR tubes (Starlabs). Cycles were designed according to standard practice, with initial 5 min denaturation at 95 C, annealing for 1 min at temperatures set according to primer Tm, and a final extension period of 7 min at 72 C.
Restriction digests were set up according to standard practice, using NheI and appropriate buffer (New England Biolabs, Ipswich, MA, USA).

Results
DNA was successfully isolated using the rapid extraction method from new and stock cultures. Three out of four low purity extracts (by A 260 /A 230 and A 260 /A 280 ratios; Table 1, Fig. 1) were of sufficient quality for PCR amplification of products of various sizes including using primers with mismatches (Fig. 2). PCR was also satisfactory from extractions from nonexponentially growing stock cultures (Supplementary Fig. S2). There was also good recovery of genomic DNA after purification, quantified by spectrophotometry (Table 2) with the desired improvement in A 260 /A 230 and A 260 /A 280 ratios in most cases (desired A 260 /A 280 of 1.8 [26]). Gel electrophoresis of all samples, with and without purification steps (Fig. 3), showed large genomic DNA fragments and no smear (Fig. 3, Supplementary Fig.  S3). Purified samples were tested in PCR as above, with all extracts now serving as templates for successful amplification (Fig. 4). This included PCR from stored (frozen) extractions ( Supplementary Fig. S3). Digests were also successful with DNA from all extracts (Supplementary Fig. S3).

Discussion
Numerous methods exist for cyanobacterial genomic DNA extraction which achieve high-quality samples suitable for sequencing. The standard use of high-quality cyanobacterial extracts, indicated by ratios of A 260 /A 230 of 2.0 and A 260 /A 280 of 1.8 [26,27], is not necessary for PCR-based cloning, screening transformants, or early investigations. Cyanobacterial colony PCR is often refractory, and material cannot be retained for future PCR reactions. Here, DNA was quickly prepared from new and longstanding Synechocystis cultures, avoiding delicate, harmful, or expensive reagents such as chloroform, lysozyme, or kit columns. The optimum density of Synechocystis cultures for rapid extraction was $8.36 Â 10 8 c.f.u./ml but this was not critical.
The simplest method provided material effective as template for PCR in the majority of cases (Fig. 2). A further purification step could achieve samples with A 260 /A 280 close to 1.8 although, even when there was little improvement in spectrophotometric purity, PCR was more successful (in the case of extract "a" which had the lowest A 260 /A 280 and a low A 260 /A 230 indicating residual carbohydrate contamination; Table 2; Figs 2 and 4). DNA visualized by gel electrophoresis revealed integrity of genomic DNA, suggesting minimal degradation. Digests were    successful on all extracts, including fresh or frozen preparations, and could be useful for library construction, for example. Therefore, this is an inexpensive and straightforward method to produce and archive genetic material, which requires minimal equipment and reagents, and can start with any extant culture of this model cyanobacterium. This should aid all early studies in Synechocystis biology and biotechnology.

Supplementary data
Supplementary data is available at Biology Methods and Protocols online.