Semi-Automated Tip Snip Cloning of Restriction Fragments into and out of Plasmid Polylinkers

Abstract

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.

Methylase-assisted subcloning for high throughput BioBrick assembly

“… The lacI-Ptac-lacO insert (A) includes a promoter that is somewhat leaky at high copy number. The IMBB2.4-pUC57-mini backbone (A–B), hereafter abbreviated pUC, is BioBrick-compatible and also includes an NsiI site downstream of PstI ( Matsumura, 2017 ). The tagRFP reporter (B) protein can cause colonies to turn visibly pink, but only when the gene encoding it is subcloned downstream of a leaky or constitutive promoter.…”

“…The insert only ligation controls produced greater background (61 ± 27 cfu/ng), which suggests that the insert was not effectively separated from the donor plasmid in this experiment. Two other established subcloning techniques, tip snip ( Matsumura, 2017 ) and 3A ( Shetty et al, 2011 ), were also used to provide standards of comparison (Table 1, also described in Supplemental Materials ).…”

“…The rhamnose promoter is regulated by catabolite repression as well as by L-rhamnose. The plasmid Prham-tagRFP-pUC ( Matsumura, 2017 ) was used to transform E. coli OmniMax 2. Limiting amounts of glucose were added to saturating concentrations of L-rhamnose (0.1%) in LB medium supplemented with ampicillin.…”

“…The lacI-Ptac-lacO-pUC donor plasmid (1µg) in 1× NEB CutSmart buffer (80 µL total reaction volume) was shortened slightly by an extra restriction enzyme (20 units PstI-HF) that recognizes a site adjacent to those used to release the insert (20 units each of EcoRI-HF and SpeI-HF) ( Matsumura, 2017 ). The tagRFP-pUC recipient plasmid (1µg) was cut as usual (20 units each of EcoRI-HF and XbaI in 1× NEB CutSmart buffer, 80 µL total reaction volume).…”

“…The p15A plasmid origin and spectinomycin resistance marker (aadA) were subcloned from pACYC Duet and pCDF Duet (EMD Millipore, Novagen) respectively into a BioBrick compatible plasmid. The intergenic region between rhaS and rhaB, which includes promoters and operators for both genes, was previously described ( Matsumura, 2017 ).…”