A one pot, one step, precision cloning method with high throughput capability

Abstract

Current cloning technologies based on site-specific recombination are efficient, simple to use, and flexible, but have the drawback of leaving recombination site sequences in the final construct, adding an extra 8 to 13 amino acids to the expressed protein. We have devised a simple and rapid subcloning strategy to transfer any DNA fragment of interest from an entry clone into an expression vector, without this shortcoming. The strategy is based on the use of type IIs restriction enzymes, which cut outside of their recognition sequence. With proper design of the cleavage sites, two fragments cut by type IIs restriction enzymes can be ligated into a product lacking the original restriction site. Based on this property, a cloning strategy called 'Golden Gate' cloning was devised that allows to obtain in one tube and one step close to one hundred percent correct recombinant plasmids after just a 5 minute restriction-ligation. This method is therefore as efficient as currently used recombination-based cloning technologies but yields recombinant plasmids that do not contain unwanted sequences in the final construct, thus providing precision for this fundamental process of genetic manipulation.

Figure 1. Cloning strategy.

Entry clone (A) and expression vector (B) are mixed in one tube together with BsaI and ligase. Of the 4 possible ligation products, A to D, only the desired product, D, is stable, while all others are redigested with BsaI. Numbers 1 to 8 denote any nucleotide of choice, and numbers in italics denote the complementary nucleotides. FOI, DNA fragment of interest.

Figure 2. Subcloning of one insert into an expression vector.

(A) Maps of entry clone pE-GFP, expression cloning vector pX-lacZ and expression construct pX-GFP. Black arrows show the position of the restriction sites for the enzymes BseRI and HindIII, and the numbers next to these indicate the sizes of the restriction fragments obtained. The grey triangle represents a Streptomyces phage C31 attB recombination site (this site is not used for the cloning procedure described here). Z, LacZ alpha fragment; N, Viral 3′ Non-translated region; T, Nos terminator; RB/LB, T-DNA right/left borders; S1–S2, selectable markers 1 and 2 (resistance to carbenicillin and kanamycin, respectively). (B) Plasmid DNA from 48 white colonies and vector digested by BseRI and HindIII and run on a 1% agarose gel. The upper and lower panels show minipreps obtained from cloning performed using ligation buffer or NEB buffer 3, respectively. DNA from all white colonies has the restriction pattern of pX-GFP (the 119 bp fragment is too faint to be visible on the picture). M: GeneRuler 1kb DNA Ladder Plus from Fermentas. V, vector pX-lacZ.

Figure 3. Subcloning of 2 and 3 inserts into an expression vector.

(A–C) Maps of expression cloning vector pX-lacZ (A), and of entry clones and the resulting constructs for 2 or 3 insert cloning experiments (B, C respectively). The positions of the restriction sites for the enzymes XmaI and BsrGI are shown as black arrows, and the expected fragment sizes indicated. (D) Restriction digest (XmaI and BsrGI) of 12 minipreps for the two insert cloning (pX-GFP-H, lane 1 to 12) and the three insert cloning (pX-S-GFP-H, lane 13 to 24), and one miniprep of the vector (pX-lacZ, lane V).

Figure 4. General strategy for generation of entry clones lacking internal BsaI sites.

For each gene of interest, two primers are designed to introduce two BsaI flanking sites (pr1, pr2), as well as one pair of primers for each internal site to eliminate (pr3, pr4). Column-purified PCR products pr1–3 and pr2–4 are mixed together with entry cloning vector (pECV) and BsaI enzyme in restriction-ligation buffer. The mix is digested for 10 minutes and heat inactivated. Ligase is then added and the mix is ligated for 10 minutes before transformation in E.coli. All white colonies contain the expected entry clone, with two flanking BsaI sites but no internal site. Horizontal arrows represent parts of the primers identical to the target sequence. Small vertical arrows indicate the location of the introduced mutation.

Figure 5. Generation of entry clones lacking internal BsaI sites for genes ycf4 and psbC.

(A) Three PCR fragments were amplified from Arabidopsis thaliana genomic (chloroplast) DNA using primers yc1–6 (gene ycf4). B, BsaI recognition sequence. The fragments were cloned in on tube into entry cloning vector pECV, resulting in entry clone pE-YCF4. Structure of the fragments for the psbC gene (not shown) is similar except that the wildtype gene contains only one internal BsaI site. (B) Restriction digest (BsaI) of minipreps from 10 white colonies for constructs pE-YCF4 and pE-PSBC. As a control, the ycf4 and psbC ORFs were amplified from genomic DNA with only the two flanking primers, and the PCR products run undigested (u, expected size 583 nt for ycf4 and 1450 nt for psbC) and digested with BsaI (d, expected sizes for ycf4: 332, 113, 54, 14, 10, expected sizes for psbC: 947, 469, 14, 10).

Figure 6. Model for subcloning of a gene of interest into a library of compatible expressions vectors.

(A) A gene of interest can be cloned as a single fragment in one entry construct (EC) or cloned as separate fragments in several entry constructs (ECs). BsaI sites 1 to 4: B1–B4. Example of three expression vectors consisting of a viral provector (V1), a TMV-based viral vector (V2) and a standard non-replicating expression vector (V3), and of the expression constructs obtained after cloning (E1–3). GOI, gene of interest; pol, TVCV RNA-dependent RNA polymerase; m, movement protein; SP, signal peptide; P, promoter. (B) Overview of the cloning strategy from entry construct to expression vector. Boxes flanking the LacZ fragment (blue) represent elements specific to the various expression vectors.