Saturation mutagenesis genome engineering of infective FX174 bacteriophage via unamplified oligo pools and golden gate assembly

Matthew S. Faber; James T. Van Leuven; Martina M. Ederer; Yesol Sapozhnikov; Zoë L. Wilson; Holly A. Wichman; Timothy A. Whitehead; Craig R. Miller

We pooled plasmid DNA containing all 14 of the phage DNA fragments (500 ng each) and digested them with BsmB1 (Fermentas Fast Digest, Life Technologies, Grand Island, NY) for 30 minutes to 1 hour at 37°C. The digested plasmids were subjected to agarose gel electrophoresis for 10 to 15 minutes using a 1.2% agarose gel to separate the vector from the inserts. The inserts were excised from the gel, purified using the GeneJET gel extraction kit (Fermentas), ligated overnight at 14°C with T4 DNA ligase (Promega Corporation, Madison, WI), and transformed by electroporation into 50 μl of XL1-Blue electocompetent cells. The transformation mix was resuspended with 950 μl of SOC and either plated immediately or incubated for up to 120 minutes (Supplemental Table S5 ) at 37°C in the presence of DNase1 (Supplemental Figure S10 ) to allow for cell lysis and the removal extracellular DNA, respectively. Aliquots of the cell lysates were added to 3 ml of ΦLB top agar containing 50 μl log-phage E. coli C cells and plated onto a ΦLB agar plate. After four to five hours of incubation at 37°C, recombinant phage plaques were visible and plates were removed from the incubator. To verify that the recombinant phage encoded the intended sequence, we picked about 30 plaques for each intended mutational target and Sanger sequenced the entire targeted gene. We also sequenced the F and G genes for about 30 wildtype plaques to assure that no mutations were naturally accumulating. Briefly, individual plaques were picked with sterile toothpicks and placed in 200 uL ΦLB and gently swirled. 1 uL of this mix was used to PCR amplify approximately ½ of the ΦX174 genome using ΦX-0F (5'-GAGTTTTATCGCTTCCATG-3') and ΦX-2953R (5'-CCGCCAGCAATAGCACC-3') primers. Internal sequencing primers ΦX-979F (5'-CGGCCCCTTACTTGAGG-3') and ΦX-1500R (5'-TTGAGATGGCAGCAACGG-3') were used to sequence gene F. ΦX-2953R was used to sequence gene G. PCR cleanups and sequencing was done at Eurofins Genomics. PCR reaction conditions were: 5 uL10X Taq buffer, 2.5 uL 10 uM ΦX-0F primer, 10 uM ΦX-2953F primer, 0.8 uL 12.5 uM dNTPs, 0.5 uL Taq polymerase (NEB #M0273), 1 uL template, 37.7 uL H20. Thermocycling conditions were 1 cycle at 95°C for 2 min, 30 cycles at 95°C for 15 sec, 52°C for 30 sec, 68°C for 2 min, 1 cycle at 68°C for 5min.