Semi-automated Tip Snip cloning of restriction fragments into and out of plasmid polylinkers<\/p>\n<\/p>\n
Emory University School of Medicine, Department of Biochemistry, O. Wayne Rollins Research Center, Atlanta, GA <\/p>\n
BioTechniques, Vol. 62, No. 3, March 2017, pp. 99106 <\/p>\n
Supplementary Material <\/p>\n
Abstract <\/p>\n
Synthetic biologists rely on semi-synthetic recombinant plasmids, but DNA synthesis is constrained by practical limits on length, accuracy, and sequence composition. Cloned DNA parts can be assembled into longer constructs via subcloning, but conventional methods are labor-intensive. One-pot recombination reactions are more convenient but harder to troubleshoot, and those that depend on PCR to create fragments with compatible ends necessitate re-sequencing. The Tip Snip protocol described here enables the subcloning of an insert from one plasmid polylinker into another without PCR or gel purification steps. Cohesive ends of unwanted restriction fragments are snipped off by additional restriction endonucleases. The resulting short fragments (snippets) are eliminated by hybridization to complementary oligonucleotides (anti-snippets) and subsequent size-selection spin-column chromatography. Unwanted linear donor vectors are ligated to double-stranded oligonucleotides (unlinkers) so that only the desired insert and recipient plasmid form circular DNA capable of transforming bacteria. This new method is compatible with high-throughput processing and automated liquid handling, and because no specialized vectors, reagents, selection schemes, or analytical techniques are required, the barriers to adoption are low. <\/p>\n
DNA synthesis costs are decreasing (1), but the assembly and cloning of synthetic DNA remains relatively labor-intensive and expensive. Nucleoside phosphoramidites are chemically synthesized on large scales. Single-stranded oligonucleotides and double-stranded synthetic genes are custom manufactured by machines, so turnover is rapid, throughput is high, and production costs are relatively low. Automation and miniaturization have decreased the per-unit cost of synthesizing gene-length (2 kb) DNAs, but further innovations are required to overcome practical limitations in length, nucleotide composition, accuracy, and yield (1). It is not yet feasible to have every new construct synthesized with its vector de novo, so PCR products and synthetic genes are most often cloned into plasmids and then sequenced. <\/p>\n
Cloned parts are often assembled into larger constructs by subcloning, but this classical approach is recalcitrant to automation for three reasons. First, robots that can load agarose gels and purify particular restriction fragments have not yet been invented. Furthermore, DNA purification, restriction digests, and ligation reactions arent reliably efficient, making monitoring and troubleshooting necessary. Finally, the design of cloning experiments is idiosyncratic, so the development of software algorithms that emulate decision making by experienced molecular biologists is non-trivial. The per-unit labor cost of subcloning (~10 h of labor per attempt, not including incubation times) far exceeds those of reagents (e.g., enzymes and purification kits). <\/p>\n
METHOD SUMMARY <\/p>\n
Tip Snip cloning uses restriction enzymes to shorten unwanted DNA fragments; the unwanted sticky ends are then neutralized by synthetic oligonucleotides. By eliminating the need to gel purify the desired restriction fragments, Tip Snip enables automation of the entire subcloning workflow. <\/p>\n
The high cost of molecular cloning has motivated the invention of new methods (2-5). In general, one-pot sequence-specific recombination reactions, such as those catalyzed by recombinases (e.g., Gateway cloning) (6, 7), thermostable polymerases (overlap extension PCR) (8), thermostable ligases (ligase chain reaction) (9), or combinations of exonuclease, polymerase, and ligase (Gibson assembly or ligase-independent cloning (10,11) are the most amenable to high-throughput and automated techniques (12). These protocols are less labor-intensive than traditional cloning workflows with discrete steps but are more difficult to troubleshoot. Another drawback of many seamless assembly techniques is their reliance on PCR or gene synthesis to create fragments with compatible ends. Every part must be re-sequenced each time it is seamlessly combined with another element and re-cloned (Special News Report. Weaver, J. 2015. BioTechniques. 59:II-III.), because the DNA polymerase I homologs used in PCR are three to five orders of magnitude less accurate than those responsible for in vivo plasmid replication and repair. Next-generation sequencing techniques lower per-unit cost, but they cannot be applied to individual plasmids. Thus, many synthetic biologists continue to assemble parts by manual subcloning. <\/p>\n
I therefore sought a way to automate the conventional restriction endonuclease\/T4 DNA ligasedependent subcloning workflow. Golden Gate assembly, which utilizes type IIS restriction endonucleases (13), and 2ab assembly (14), which utilizes plasmids with two selectable markers separated by a unique restriction site, obviate gel purification but necessitate the employment of specialized vectors incompatible with those of other cloning standards. The three antibiotic assembly (3A) protocol (15) was specifically designed to assemble parts compatible with the seminal BioBrick Assembly Standard (RFC10) used by many synthetic biologists (16). Two donor plasmids carrying parts and a recipient plasmid encoding a counter-selection marker along with a selectable marker different than those of the donors are digested with different pairs of restriction enzymes. All six of the resulting restriction fragments are ligated together and used to transform Escherichia coli. The desired recombinant construct is distinguished from the parental plasmids using an antibiotic and the counter-selection scheme. This technique circumvents gel purification, but sacrifices efficiency for convenience. Three-fragment ligations dont occur as frequently as two-fragment reactions, particularly when three other unwanted fragments with compatible cohesive ends are present. The extraneous DNA also inhibits heat shock transformation of chemically competent E. coli (17), and electroporation is sensitive to salts in ligation reactions so it is less amenable to high-throughput experiments. Here, I describe a set of expedients that in combination facilitate efficient and reliable cloning of DNA into or out of almost any existing plasmid polylinker (multiple cloning site) without the need for PCR amplification or gel purification. Materials and methods <\/p>\n
The approach described here builds upon the following classical cloning techniques (18), except as noted. A more detailed step-by-step protocol is included in the Supplementary Material. Plasmids were purified from transformed E. coli using silica spin columns (QIAGEN, Valencia, CA) and hydrated Sephadex G-50 (GE Healthcare Life Sciences, Pittsburgh, PA) in empty spin columns (Epoch Life Science, Missouri City, TX) as directed by their manufacturers. Restriction digests were set up as recommended by the supplier [New England BioLabs (NEB), Ipswich, MA]. Whenever possible 2 g of DNA (6 nM for a 5 kb plasmid) were digested to completion (or nearly so) at 37C overnight with 2040 U of each restriction enzyme (12 nM) in 100 L total reaction volume. Approximately 20 fmol of digested, purified recipient plasmid and 20 fmol of digested insert (and donor plasmid) were reacted with 0.31 Weiss units of T4 DNA ligase in NEB T4 DNA ligase buffer containing 5% polyethylene glycol (molecular weight: 8000) (19). The reactions were temperature cycled in a Bio-Rad (Hercules, CA) MJ mini thermocycler between 10C for 30 s and 30C for 30 s for a total of 418 h (20). <\/p>\n
Chemically competent E. coli OmniMax2 cells (Thermo Fisher Scientific, Waltham, MA) were prepared according to Inoue et al. (17). For each transformation, up to 1.25 ng total DNA in ligation reactions were used to transform 25 L of competent cells in the thermocycler. The transformants were spread on lysogeny broth medium (LB) agar plates containing 100 g\/mL ampicillin. Some agar plates also contained inducer and a histochemical substrate as described below. Some colonies were adsorbed to a nitrocellulose filter and transferred colony-side up to fresh LB-ampicillin plates supplemented with inducer (1 mM IPTG, 10 g\/mL tetracycline, 0.4% L-arabinose or 0.4% rhamnose) and 2 mg\/25 mL plate X-gal (for colonies carrying lacZ expression vectors). Additional information the reagents and materials used to culture the bacteria can be found in the Supplementary Material. Results and discussion <\/p>\n
The Tip Snip cloning workflow begins with plasmids prepared via alkaline lysis and silica spin-column chromatography. A QIAcube robotic workstation (QIAGEN) can be used to automate this and other purification protocols. To eliminate small molecules that might inhibit restriction endonucleases or broaden their sequence specificity (21), plasmids are further purified via manual gel-filtration spin-column chromatography. Recipient and donor plasmids are digested as usual with restriction enzymes that produce fragments with compatible cohesive ends. Additional restriction enzymes recognizing sites in the polylinker are used to shorten undesired restriction fragments (the stuffer fragment of the recipient plasmid or the donor plasmid of a subcloning experiment) (Figure 1). <\/p>\n
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