Flp variant gene libraries were generated by shuffling the genes of several target-specific Flp variants: FV79 (link), FV-2798-7, FV61-2, FV-63-n36, FV-71-6017 (link), and Flp-sup337 (link). The gene mixture also contained the wild-type Flpe gene50 (link) and the Flpe gene library with the randomized codons at positions 55, 58, and 59. Site-specific mutagenesis to randomize Flp codons at positions 55, 58 and 59 was performed as describe earlier50 (link) using oligonucleotides that contained all ‘N’s at the respective positions.
The DNA shuffling was performed essentially as described in Bolusani et al.9 (link). In brief, the Flp variant genes were first amplified using Taq polymerase (New England Biolabs) and then the resultant PCR products were mixed and fragmented with DNase I. These DNA fragments were reassembled into the Flp gene library by amplifying them first using Pfu–Ultra polymerase (Agilent, Santa Clara, CA) without any specific primers and then by amplifying the resultant PCR products using Taq polymerase with primers that anneal just outside the coding region of the Flp gene. The PCR products of the second amplification (that contained the Flp variant libraries) were cloned into the inversion reporter.
The DNA shuffling was performed essentially as described in Bolusani et al.9 (link). In brief, the Flp variant genes were first amplified using Taq polymerase (New England Biolabs) and then the resultant PCR products were mixed and fragmented with DNase I. These DNA fragments were reassembled into the Flp gene library by amplifying them first using Pfu–Ultra polymerase (Agilent, Santa Clara, CA) without any specific primers and then by amplifying the resultant PCR products using Taq polymerase with primers that anneal just outside the coding region of the Flp gene. The PCR products of the second amplification (that contained the Flp variant libraries) were cloned into the inversion reporter.
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