MAGE-Seq of Escherichia coli dihydrofolate reductase
The EcNR2 strain derived from E. coli K12 MG1655 was used to perform competition experiments between the wild type strain and all 63  mutants at each codon of the folA gene. Mutagenesis was performed using Multiplex Automated Genomic Engineering (MAGE). 90 bp DNA oligomers were designed around each folA codon using the MG1655 wild type sequence as reference. For each codon, all possible nucleotide variants were synthesized. To avoid simultaneous mutations of multiple codons, cells were transformed targeting ten consecutive codons at a time. After four rounds of electroporation, cells were recovered and pooled together at approximately the same concentration based on cell counts. Two competition growth experiments were carried out, one for each half of the protein. For the competition experiments, cells were grown in LB media in a turbidostat while maintaining constant volume and cell density. Samples were taken at least every 2 hours for a period of 18 to 24 hours, spun down, washed in PBS, spun down again and stored at -20C until all samples were collected. For each competition, the corresponding folA region was amplified through PCR while assigning a specific DNA barcode for each time point. PCR products were then pooled and paired-end sequenced using the MiSeq Reagent Kit 2 from Illumina.
dhfr2.7