Pfu ultra polymerase

pcDNA5/FRT/TO (Invitrogen, Carlsbad, CA) containing IRE1α-HA, wIRE1α (IRE1α-∆434–443 HA) and IRE1α-K907A-HA were described previously (Plumb et al., 2015 (link)). IRE1α-T446A-S450A-T451A-HA mutant was created using previously described primers (Sun et al., 2015 (link)). sIRE1α (IRE1α-S439A-T446A-S450A-T451A-HA), IRE1α-V437A-D443A-HA, IRE1α-∆434-443A-K907A-HA, in pcDNA5/FRT/TO were made by site-directed mutagenesis. Prl-His-2xstrep-IRE1α-FLAG constructs were generated by first inserting Prl-His-2xstrep into pcDNA5/FRT/TO using standard methods. Next, IRE1α-FLAG was amplified beginning from amino acid 29 and cloned into pcDNA5/FRT/TO Prl-His-2xstrep. Mouse spliced XBP1 plasmid (Addgene# 21833) is a kind gift from Dr. David Ron. All PCR reactions were performed with Phusion high fidelity DNA polymerase (NEB, Ipswich, MA), except for site directed mutagenesis, which used Pfu-Ultra polymerase (Agilent technologies, Santa Clara, CA). 3% DMSO was included in all PCR reactions to enhance amplification. The coding regions of all constructs were sequenced to preclude any sequence error. The Yale Keck DNA Sequencing Facility performed all sequencing services.

All chemicals were reagent or molecular biology grade. Pfu ultra polymerase (600380–51) was purchased from Agilent. 6x DNA loading dye (R0611), 1kb DNA ladder (SM1333), and 10 mM premixed dNTPs (R0192) were purchased from Thermo-Scientific. Phusion polymerase (M05305), BamHI-HF (R31365), NDE1 (R011S), and Gibson Assembly Master Mix (M5510A) were purchased from New England Biolabs. QIAquick gel extraction kit (28706) and QIAprep spin miniprep kit (27004) were manufactured by Qiagen. All oligonucleotides for PCR amplification and mutagenesis were purchased from IDT. ATP (987-65-5) was purchased from Pharma Waldhof. UDP (94330), and UMP (U6375) were purchased from Sigma-Aldrich. D-Luciferin (14681) was made by Cayman Chemical.

Sequences encoding full-length human RIG-I or MDA5 with N- or C-terminal FLAG/HA-tag were cloned into pcDNA5 FRT/TO (Invitrogen, Carlsbad, CA, USA). Mutants were generated by site-directed mutagenesis with PfuUltra polymerase (Agilent, Santa Clara, CA, USA).

His-tagged pol V was purified from E. coli stain RW644 as described by Karata et al. (2012) (link) and RecA WT was purified by a standard protocol (Cox et al., 1981 (link)). RecA E38K/K72R and RecA E38K/ΔC17 were provided by Michael Cox at the University of Wisconsin, Madison. We incorporated p-azido-L-phenylalanine (pAzF) (Chin et al., 2002 (link)) into RecA WT to site specifically label the protein with Alexa Fluor 488 DIBO alkyne. For the cloning of RecA, we used the pAIR79 plasmid, which was a gift from Michael Cox at the University of Wisconsin, Madison. The Phe21 sequence in RecA WT was replaced with the amber codon via site-directed mutagenesis using Pfu Ultra polymerase (Agilent Technologies). Once the sequence was confirmed, pAIR79 was cotransformed with the vector pEVOL-pAZF (a gift from the Peter Schultz lab at The Scripps Research Institute, San Diego, CA) into the BLR expression strains (Young et al., 2010 (link)). The RecA_F21AzF protein was purified using the same standard protocol for RecA WT (Cox et al., 1981 (link)). RecA_F21AzF was labeled with Alexa Flour 488 (RecA_F21AzF-Alexa Fluor 488) according to manufacturer's instructions (Life Technologies).

Plasmid DNA from bacterial cells was isolated using E.Z.N.A. Plasmid Mini Kit I (Omega Bio-tek). Genomic DNA from mammalian cells was isolated using E.Z.N.A. Tissue DNA Kit (Omega Bio-tek). Amplification of the DNA fragments for cloning was performed using Pfu-Ultra polymerase (Agilent). PCR analysis of the genomic DNA was performed using Q5 (New England Biolabs) and GoTag (Promega) master mixes. The ClustalW program (https://www.genome.jp/tools-bin/clustalw) was used for sequence alignments. Golden Gate TALEN kit was used to assemble the DNA binding domains of TAL^{28 (link)}. General genetic engineering experiments were performed as described in Molecular Cloning Manual⁵¹ . 3D structures of the proteins were analyzed using Swiss-PdbViewer^{52 (link)}.

Flp variant gene libraries were generated by shuffling the genes of several target-specific Flp variants: FV7^{9 (link)}, FV-2798-7, FV61-2, FV-63-n36, FV-71-60^{17 (link)}, and Flp-sup3^{37 (link)}. The gene mixture also contained the wild-type Flpe gene^{50 (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 earlier^{50 (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.