Bioproduction of pure, kilobase-scale single-stranded DNA

Abstract

Scalable production of kilobase single-stranded DNA (ssDNA) with sequence control has applications in therapeutics, gene synthesis and sequencing, scaffolded DNA origami, and archival DNA memory storage. Biological production of circular ssDNA (cssDNA) using M13 addresses these needs at low cost. However, one unmet goal is to minimize the essential protein coding regions of the exported DNA while maintaining its infectivity and production purity to produce sequences less than 3,000 nt in length, relevant to therapeutic and materials science applications. Toward this end, synthetic miniphage with inserts of custom sequence and size offers scalable, low-cost synthesis of cssDNA at milligram and higher scales. Here, we optimize growth conditions using an E. coli helper strain combined with a miniphage genome carrying only an f1 origin and a β-lactamase-encoding (bla) antibiotic resistance gene, enabling isolation of pure cssDNA with a minimum sequence genomic length of 1,676 nt, without requiring additional purification from contaminating DNA. Low-cost scalability of isogenic, custom-length cssDNA is demonstrated for a sequence of 2,520 nt using a bioreactor, purified with low endotoxin levels (<5 E.U./ml). We apply these exonuclease-resistant cssDNAs to the self-assembly of wireframe DNA origami objects and to encode digital information on the miniphage genome for biological amplification.

Figure 1

Scalable bacteriophage production of isogenic cssDNA. (a) Miniphage phPB52 was assembled using restriction-free (RF) cloning was used for miniphage phPB52 assembly and transformed into E. coli containing the M13cp helper plasmid for production of isogenic cssDNA. (b) aPCR was used to generate the two ssDNA megaprimers for RF cloning encoding the f1 origin of replication (f1 ori) and the bla ampicillin selection marker (Selection). See Fig. S1a for uncropped color image. (c) Phage particles from clarified media were visualized by TEM. See Fig. S1b for uncropped image. (d) DNA purification from the bacterial pellet and the clarified media show mostly pure cssDNA in the media and cssDNA and dsDNA phagemid, and helper plasmid in the bacterial pellet. See Fig. S1c for uncropped color image.

Figure 2

Shaker flask production of pure cssDNA. (a) Shaker flask growth of phPB84 was used to optimize conditions for phage amounts and purity. (b) Time-course assay of cssDNA production of phPB84, with cssDNA yield calculated by absorbance at 280 nm and purity adjusted by agarose gel band intensity, showing maximum yield and purity at the 8-hour timepoint. The 16 h time-point is from a separate culture, and therefore is not included in the plot. See Fig. S1a for uncropped color gel image. (c) Comparison between DH5a F′Iq and SS320 showing two-fold yield increases in the SS320 strain. (d) Comparison between growth media showing five-fold improved cssDNA yield in 2 × YT after 8 hours of production. (e) Comparison of five pH values for cssDNA production, controlled by use of 100 mM HEPES-NaOH. Error bars indicate standard deviation of triplicate experiments. See Figs S3 and S3 for uncropped color triplicate measurements of all experiments.

Figure 3

Batch fermenter production of pure cssDNA. (a) Scalable production in a stirred-tank bioreactor. (b) Time-course assay of cssDNA yield based on agarose gel band intensity analysis (Fig. S5), with the 8-hour timepoint used for 900 mL cssDNA purification. (c) Silica-column DNA purification from the PEG-precipitated phPB84 phage showed no detectible dsDNA contamination, similar in purity to commercially available M13mp18, yielding 2 mg of DNA per liter of culture at the 8-hour timepoint. Stability from exonuclease I (ExoI) degradation after 30 min coincubation indicates the ssDNA is circular.

Figure 4

Applications of scalable, isogenic miniphage production. (a) Pentagonal bipyramids of 52-bp and 84-bp edge-lengths were folded using the phPB52 and phPB84 as scaffolds, respectively. Agarose gel shift mobility assays and TEM were used to validate the folding of the scaffold to the expected design. See Figs S7 and S8 for uncropped gel images and example full field TEM micrographs, respectively. Scale bars represent 20 nm. (b) Phage particles are natively protected from environmental degradation and easy to amplify by bacterial infection, and thus provide a compelling method for archival and amplification of digital information encoded in DNA. The encoding scheme shown here can generate bio-orthogonal sequences that are designed to limit secondary structure and recombination sites. DNA encoding a digital text file containing a line from The Crucible (full encoded text: “The answer is in your memory and you need no help to give it to me. Why did you dismiss Abigail Williams?”) was ligated to the phPB52 vector and subsequently produced and amplified in bacteria. Sanger sequencing by primer walking was used to retrieve the original digital file.