For hybridization during recombination experiments, a constant molar ratio of 1:4 [fragments A1, B1, and C1; each 0.09 pmol and each ~5.4 kB in size, including the linearized pET22b(+) vector, to 12 fragments: A2–A5, B2–B5, and C2–C5; each 0.36 pmol and each 0.17–0.45 kB] proved to be best to ensure hybridization (Figure 1). The molar ratio of 0.09 pmol to 0.36 pmol also resulted in good recombination and transformation results (see the Results and discussion section). After iodine cleavage, all 15 DNA fragments (five domains of each of the three phytase genes) were pooled on ice (Figure 1) and subsequently heated (70°C for 3 min; Eppendorf Mastercycler) to avoid preferential hybridization due to the order in pipeting. After incubation at room temperature (20°C for 5 min), 3 μL hybridization reaction were transformed without further purification or ligation into chemically competent E. coli BL21 Gold (DE3) cells (50 μL) following a standard protocol (28). Transformation mixtures were spread on agar plates with Luria Bertani (LB) medium supplemented with 100 mg/mL ampicillin and incubated overnight at 37°C.
Colonies carrying the pET22b(+) vector with shuffled phytase domains were directly transferred into 96-well master plates, grown overnight in 100 μL LB medium with 100 mg/mL ampicillin at 37°C, and stored at -80°C after the addition of 100 μL (50% vol) glycerol. Ninety-six–well plates containing solid agar (GATC Biotech, Konstanz, Germany), were inoculated with clones harboring the pET22b(+) vector with shuffled phytase chimeras and sent to GATC Biotech for sequencing (oligonucleotides: P33, P34; Supplementary Table S1). Clone Manager 9 Professional Edition software (Scientific & Educational Software, Cary, NC, USA) was used for analyzing sequencing data.Results and discussion
Three phytases, appA from Escherichia coli, ympH from Yersinia mollaretii, and phyX from Hafnia alvei were chosen to serve as model genes for development and validation of the PTRec method. The number of crossover points was set to four within the targeted genes, since most proof of concept demonstrations of gene shuffling methods have been performed with one to four crossover points. PTRec comprises four steps: (1) DNA fragment amplification by PCR using primers with complementary phosphorothioate nucleotides at the 5'-end; (2) chemical cleavage of the PCR products in an iodine/ethanol solution for generating single-stranded overhangs (12 nucleotides); (3) random DNA fragment assembly to full-length chimeric genes through hybridization at room temperature; and (4) transformation into competent host cells without further DNA purification or enzymatic DNA-ligation (Figure 1).
As a preparatory step, all phytases were subcloned for domain recombination experiments into the pET22b(+) vector by PLICing. The vector backbone together with the N-terminal portion of the genes was regarded as domain A1, B1, and C1, respectively, for the three phytase genes (Figure 1). A protein sequence alignment of the three phytases was used to select four crossover sites for recombination (Supplementary Figure S1). Identical stretches of four amino acids at potential crossover sites are necessary to design gene-specific complementary oligonucleotides with 12 phosphorothioate nucleotides at the 5'-end (Supplementary Table S1 and Supplementary Figure S1). One additional crossover site near the stop codon was chosen to recombine five protein domains of the three phytases in total (Figure 1). The 15 DNA fragments for domain shuffling were generated and purified as described in the Material and methods section (Figure 1, step 1). PTRec relies on the specific hybridization of single stranded 5'-ends of multiple DNA fragments and a vector backbone as described for the PLICing method (24) and the OmniChange method (25), which were developed as a cloning technology and a method for simultaneous site-saturation of five codons, respectively. In PTRec, overhangs are generated by chemical cleavage of the phosphorothioate bonds with iodine in an alkaline ethanol solution (Figure 1, step 2) (23). Phosphorothioate bonds were incorporated into the DNA fragments during PCR amplification using primers with 12 consecutive phosphorothioated nucleotides at the 5'-end (Supplementary Table S1 and Supplementary Figure S1). Critical for random but oriented assembly of the five fragments was a heat incubation protocol, which enables oriented hybridization of all DNA fragments to full plasmid size (Figure 1, step 3). The protocol ensures an efficient and stable assembly of phytase chimeras consisting of five fragments and 10 nicked DNA positions, even under transformation conditions. The latter could be achieved by heat incubation at 70°C for 3 min in the PCR-Cycler, cooling to 20°C for 5 min in the PCR-Cycler, and subsequent incubation on ice until transformation, which yielded 1100 clones (transformation efficiency: 5.1 × 103 cfu/μg DNA).