2University of Ljubljana, Faculty of Pharmacy, Ljubljana, Slovenia
The growing popularity of the lactic acid bacterium Lactococcus lactis has increased demand for novel high-throughput cloning methods. Here we describe a general TA-cloning methodology and demonstrate its feasibility using the plasmid pNZ8148. PCR products were directly ligated into a linear, PCR-amplified and XcmI-digested pNZ8148 derivative that was termed pNZ-T. Cloning using pNZ-T yielded a high proportion of insert-containing plasmids on transformation. Although demonstrated with L. lactis, the technique presented here is organism-independent and can be implemented in other plasmids.
Lactococcus lactis is a Gram positive lactic acid bacterium with industrial importance. Genetically modified derivatives of L. lactis have been tested for delivery of therapeutic proteins to the gut (1), and it is also increasingly popular for expression of membrane proteins (2). Commercially available cloning and expression systems for L. lactis are sparse, and largely inferior to those of established systems such as Escherichia coli, Saccharomyces cerevisiae, and mammalian cell lines, and cloning is currently based mainly on a classic restriction enzyme digest, which is not amenable to high-throughput applications. Therefore, new methods for rapid and restriction enzyme independent cloning of selected L. lactis genes are being sought. Ligation independent cloning was reported, but with the use of an intermediary E. coli plasmid (3). Recently, a Gateway cloning system was introduced as well (4). Here we describe the development of a TA-cloning system based on the nisin-controlled expression system (NICE) plasmid pNZ8148 (5). TA-cloning plasmids contain a single thymidine residue at both 3'-ends of the linearized plasmid. This 3'-thymidine is compatible with Taq DNA polymerase-amplified PCR products, which contain 3'-adenosine residues due to terminal transferase activity of the polymerase (6). Several TA-plasmids for cloning in E. coli are commercially available (pGEM-T from Promega, Madison, WI, USA, and TOPO TA-cloning kits from Invitrogen, Carlsbad, CA, USA). TA-plasmids are currently produced by either blunt restriction digest of a plasmid followed by addition of T-overhangs using Taq DNA polymerase or terminal deoxynucleotidyl transferase (7) or by using restriction enzymes that create single 3'-overhangs (e.g., XcmI) (8). Restriction digestion with XcmI often yields only partial cleavage, resulting in a relatively high background of empty plasmids when cloning (9). In addition, the use of XcmI for TA-plasmid preparation usually involves introducing an XcmI-containing cassette (9, 10), which limits flexibility in terms of the site of the PCR product integration and requires prior plasmid modification.
Our approach is based on whole plasmid PCR amplification with a high fidelity and processivity polymerase (e.g., KOD DNA polymerase; Toyobo Novagen, Osaka, Japan). Two primers, both containing the XcmI recognition site, are designed to target the region where insertion of the PCR product is desired. This methodology enables PCR products to be cloned at a custom site on the plasmid, and requires no prior plasmid modification (Figure 1). We demonstrate the feasibility of our strategy by preparing pNZ-T, a derivative of pNZ8148 that enables cloning of PCR products downstream of the nisA promoter, immediately following the ATG start codon. The cloned gene can therefore be expressed on the addition of nisin (5). Additionally, the reverse primer was designed to introduce the NdeI restriction site sequence downstream of the cloned gene, which enables further subcloning of the gene through NcoI/NdeI restriction.
pNZ-T was amplified from pNZ8148 using pNZ1 (5'-TTACCATATCACCCATGGTGAGTGCCTCC-3') and pNZ2 (5'-AATCCAAGACACATATGGCTTGAAACGTTCAATTGAAATG-3') primers. KOD DNA polymerase (Toyobo Novagen) was used according to the manufacturer's recommendations. Twenty-five cycles of PCR were performed as follows: denaturation at 95°C for 20 s, annealing at 50°C for 10 s, and extension at 70°C for 75 s. The amplification product (Supplementary Figure S1) was cut from an agarose gel, purified with Wizard SV Gel and PCR Clean-Up System (Promega), and then digested with 10 U XcmI (New England Biolabs, Beverly, MA, USA) at 37°C for 16 h. This was followed by an optional step, in which KpnI and XbaI (10 U each; both from New England Biolabs) were added to the mixture and incubated for a further 60 min to degrade any remaining pNZ8148 template. Following restriction digestion, the DNA was again purified using Wizard SV Gel and PCR Clean-Up System (Promega) to yield pNZ-T.
Test PCR inserts were amplified with DreamTaq Green DNA Polymerase (Fermentas, St. Leon-Rot, Germany), which creates A-overhangs. Primers and templates from our laboratory collection were used to prepare amplicons of various sizes (924, 516, and 108 bp; Supplementary Figure S1), which were gel-purified as described above. Alternatively, high-fidelity DNA polymerases, which do not create A-overhangs, could be applied, using the A-tailing procedure (described in instruction manuals of commercial TA-cloning systems, e.g., pGEM-T; Promega).
pNZ-T (200 ng) and 1–2 ng/bp of insert were added to the ligation mixture in a total volume of 20 µL with T4 DNA ligase (Promega). The ligation reaction proceeded at 16°C for 19 h.
Ethanol-precipitated plasmid was introduced into L. lactis NZ9000 cells by electroporation according to Holo and Nes (11), using a Gene Pulser II apparatus (Bio-Rad, Hercules, CA, USA). Bacteria were spread on agar plates containing M-17 medium (Merck, Darmstadt, Germany) supplemented with 0.5% w/v glucose (GM-17) and10 µg/mL chloramphenicol. Plates were incubated at 30°C for 2 days.
We used colony PCR to test the bacterial colonies for the presence of insert-containing pNZ-T. Colonies were transferred to a PCR mixture without Taq DNA polymerase and incubated at 99°C for 10 min to release the DNA. This was followed by 30 cycles of PCR as follows: denaturation at 95°C for 30 s, annealing at 46°C for 1 min, and extension at 72°C for 1 min. DreamTaq Green DNA Polymerase (Fermentas), with corresponding reagents, was applied for the amplification, together with primers NZ-Uni (5'-GTTAGATACAATGATTTCG-3') and NZ-Rev (5'-TGCTTTTTGGCT-ATCAATC-3'). At least 30 colonies were tested for each cloned fragment. The percentage of insert-containing clones and the transformation efficiency were calculated (Table 1). The low level of empty plasmid obtained represents a substantial advance upon previous TA-cloning approaches and is the consequence of PCR amplification of the plasmid. Self-ligation of the PCR product is not favored, thus minimizing the problem of partial XcmI digestion that was responsible for the high background of empty plasmids reported previously (8). A similar efficiency (above 90%) was obtained without KpnI /XbaI digestion of the template plasmid.
Plasmids from several colonies were isolated with Wizard SV Minipreps (Promega), using an additional lysozyme treatment prior to the cell lysis step, and the correct processing of the plasmid with XcmI and integration of the Taq DNA polymerase-generated PCR fragments was verified by nucleotide sequencing (Eurofins MWG Operon, Ebersberg, Germany).
We have developed a TA-cloning plasmid, pNZ-T, based on the plasmid pNZ8148 that should be useful for researchers working with L. lactis, since the NICE system is well established in L. lactis for recombinant protein expression. Direct cloning of PCR products into pNZ-T is not only time- and cost-effective, but also highly appropriate for high-throughput cloning of genes for expression in L. lactis, since it requires no intermediate plasmid host. In addition, our basic methodology for preparing TA-cloning plasmids is an improvement over existing ones. It includes whole-plasmid PCR amplification, which increases the percentage of colonies with insert-containing plasmid upon transformation and offers greater flexibility since, with appropriate primer design, the TA-cloning site can be introduced at any position on the plasmid, including regions where no restriction sites are available that may prove suitable for testing plasmid elements. For these reasons, this technique could find widespread use, as it can be applied to other plasmids from other organisms.The size of the plasmid that can be amplified is determined by the properties of the DNA polymerase used. KOD DNA polymerase can, according to the manufacturer, amplify DNA fragments up to 6000 bp in length, which limits the upper size of the plasmid. It should be noted that introduction of a custom T-overhang site requires consideration of an additional TA base pair, which should be taken into account during primer design to prevent issues such as frame shifts. In conclusion, our improved cloning methodology is easy to use and should prove adaptable for researchers in other fields thereby stimulating the custom use of TA-cloning.
This study was supported by the Slovenian Research Agency grant no. P4-0127. We are grateful to Prof. Roger Pain for critical reading of the manuscript.
The authors declare no competing interests.
Address correspondence to Aleš Berlec, Department of Biotechnology, Jožef Stefan Institute, Jamova 39, SI-1000 Ljubljana, Slovenia. Email: firstname.lastname@example.org
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