All chemicals were analytical grade and purchased from Sigma-Aldrich (Steinheim, Germany), Serva (Heidelberg, Germany), or AppliChem (Darmstadt, Germany). Enzymes were obtained from Fermentas (St. Leon-Rot, Germany), and all oligonucleotides were purchased from Eurofins MWG Operon (Ebersberg, Germany). Oligonucleotides were diluted in Milli-Q water (Millipore, Billerica, MA, USA) to a final concentration of 100 μM. All oligonucleotides used in this study are summarized in Supplementary Table S1. The genes of the three phytases—appA of Escherichia coli (GenBank, AF537219), ympH of Yersinia mollaretii (GeneBank, JF911533), and phyX of Hafnia alvei (Patent, U.S. no. 2008/0263688 A1)—were used for recombination as proof of concept for the PTRec method. The expression vector pET22b(+) was obtained from Novagen (Merck KGaA, Darmstadt, Germany), and E. coli BL21-Gold (DE3) was purchased from Invitrogen (Karlsruhe, Germany). Chemically competent E. coli BL21-Gold (DE3) cells (transformation efficiency: 1.4 × 106 cfu/μg pUC19) were prepared according to a published procedure (26).PCR amplification of phytase genes, individual phytase domains, and vector pET22b(+)
DNA isolations were performed using QIAprep Spin Miniprep kit (Qiagen, Hilden, Germany). PTRec requires only a short stretch of four amino acids (AS) that is identical among the proteins to be recombined in order to define a single crossover point for recombination. In the three model phytases, four crossover points were identified by multiple protein sequence alignments, giving rise to five different protein domains varying in size (domain A, ~50 amino acids; domain B, ~105 amino acids; domain C, ~155 amino acids; domain D, ~50 amino acids; and domain E, ~55 amino acids). As a hybridization method, PTRec requires sequence identity on the nucleotide level at the respective crossover points (see Supplementary Table S1). Despite identity on the protein level, the short crossover point stretches showed diversity on the nucleotide level due to the degeneracy of the genetic code. Therefore, 35 silent mutations in total were introduced into 19 out of 30 primers during oligonucleotide design. PCRs were performed in a final volume of 50 μL in a Gradient Cycler (Eppendorf, Darmstadt, Germany) using thin-wall PCR tubes (Sarstedt, Nuembrecht, Germany). PCRs for subcloning purposes were composed of 1× PfuS buffer, 0.2 mM dNTP mix, 400 nM forward and reverse primer (appA, P1 and P2; ympH, P3 and P4; phyX, P5 and P6; see Supplementary Table S1), 5 U PfuS DNA polymerase, and 20 ng template DNA. PCR cycling conditions: initial denaturation at 94°C for 2 min, 35 cycles [94°C for 30 s, 55°C for 35 s, 68°C for 50 s (phytases) or 4 min (pET22b(+)], and one fill up cycle (68°C for 10 min). Before iodine cleavage, the PCR products were DpnI-digested to remove the template DNA, column-purified using a PCR purification kit (Macherey-Nagel, Dueren, Germany), and quantified using a NanoDrop 1000 UV spectrophotometer (NanoDrop Technologies, Wilmington, DE, USA). For amplification of individual phytase domains, PCRs contained 1× Taq buffer, 0.2 mM dNTP mix, 400 nM forward and reverse primers (see Supplementary Table S1), 5 U Taq DNA polymerase, and 40 ng of the respective pET22b(+) vector templates. PCR cycling conditions: initial denaturation at 94°C for 2 min, 35 cycles [94°C for 30 s, 55°C for 30 s, 72°C for 7 min (domains A1, B1, and C1, which include the pET22b(+) vector sequence; see Figure 1], or 1 min (all domains 2–5; see Figure 1) and one fill-up cycle (72°C for 5 min). Following completion of the PCRs, DpnI was added to remove template DNA. Subsequently, PCR products were purified and quantified (NanoDrop). Agarose-TAE gel electrophoresis was performed according to a standard protocol (27) to confirm presence and size of amplified genes, individual domains, or vectors. Colony PCRs were carried out before sequencing, following the subcloning PCR protocol with only slight modifications: a master mix was dispensed into 20-μL aliquots in PCR tubes. Colonies were directly transferred into PCR tubes with a sterile pipet tip. Initial denaturation time at 94°C was increased to 5 min to ensure cell lysis, and 5 μL the resulting PCR products were analyzed by agarose gel electrophoresis.
Iodine treatment, DNA-fragment hybridization, and transformation
For recombination purposes, iodine treatment and DNA fragment hybridization procedures were optimized to recombine multiple DNA fragments using the phosphorothioate cleavage principle reported by Blanusa et al. (24). Finally, a mixture of 4 μL DNA (0.03–0.6 pmol), 0.5 μL cleavage buffer (0.5 M Tris-HCl, pH 9.0), 0.3 μL iodine stock solution (100 mM iodine in 99% ethanol), and 0.2 μL Milli-Q water proved to be best for DNA cleavage. Samples were incubated in PCR tubes (70°C for 5 min; Eppendorf Mastercycler) and kept on ice until further use. Generated DNA fragments were subsequently hybridized without any purification by carefully mixing with a pipet. For phytase subcloning, 0.15 pmol cleaved pET22b(+) and 0.2 pmol cleaved appA, 0.22 pmol ympH, or 0.25 pmol phyX were combined. Resulting mixtures were incubated at 20°C for 5 min prior to transformation.