Protein Engineering Protocols - Mycobacteriology research center

Degenerate Oligonucleotide Gene Shuffling 199using the common flanking nested 5′ and 3′ primers under the following conditions:1 cycle of 95°C for 1 min to activate the enzyme; then 20 cycles at 95°C(denaturation) for 30 s; annealing at 50°C for 20 s; extension at 72°C for 40 s; anda final incubation at 72°C for 5 min, using Platinum Pfx DNA polymerase.3.1.6. Cloning of Shuffled Products1. Digest DOGS PCR products with the restriction enzymes BamHI and HindIII.2. Digest pBSII KS– with the same restriction enzymes and treat with shrimp alkalinephosphatase.3. Ligate the PCR product in the vector pBSII KS–.4. Transform the ligated plasmid, which also carries an ampicillin resistance geneinto the E. coli strain DH5α and spread onto LB plates containing 100 µg/mLampicillin and 5 mM isopropylthiogalactoside (IPTG).5. Pick individual colonies, patch in duplicate onto new plates containing 100 µg/mLampicillin and 5 mM IPTG, and screen for the expression of xylanase activity bythe Congo Red overlay method (17) as detailed in Subheading 3.1.7.3.1.7. Plate Assays Using the Congo Red Method1. Gently pipet 4 mL of overlay solution cooled to approx 50°C onto plates with patchedcolonies. The plates should be prewarmed to 37°C to ensure that the overlay solutiondoes not set before forming an even layer.Fig. 3. (Opposite page) CDE primers for PCR and overlap extension. (A) A diagrammaticrepresentation of double-stranded template DNA and the relative binding positionsof the CDE forward and reverse primers. In separate PCR amplifications, the forwardCDE primer is used combination with the reverse flanking primer, whereas the reverseCDE primer is used in combination with the forward flanking primer. (B) A diagrammaticrepresentation showing the correct binding of each of the CDE forward and reverseprimers to the DNA template. A thin vertical line (|) indicates correct primer/templatepairing of adjacent nucleotides; a colon (:) indicates potential matching of adjacentnucleotides caused by degeneracy in the primer pool; whereas a dash (–) indicates anucleotide mismatch. As depicted here, in the first round of PCR, the nondegenerate coredoes not contribute to primer binding, and primer binding specificity is attained by the 3′degenerate end of each primer. (C) A diagrammatic representation showing the bindingof each of the CDE forward and reverse primers to products generated in early rounds ofPCR amplification. A thin vertical line (|) indicates correct primer/template pairing ofadjacent nucleotides; a colon (:) indicates potential matching of adjacent nucleotidescaused by degeneracy in the primer pool. The nondegenerate core now acts as a clamp,ensuring efficient use of the degenerate primer pool in amplification of a gene segment.(D) A diagrammatic representation showing the complementarity of two gene segmentsgenerated by PCR using, respectively, the forward or the reverse CDE primer. This complementarityallows for efficient polymerase-mediated overlap extension, resulting in theregeneration of a single DNA fragment comprised of both DNA segments. If the two PCRproducts originated from different genes, a chimeric fragment will be generated.