Oligonucleotide pools

Gene blocks, oligonucleotide pools, and oligonucleotides used for 3Cs reactions, cloning, PCR, and sequencing libraries were obtained from Integrated DNA Technologies (IDT) or Merck KGaA.

To generate promoter libraries for Sort-Seq, mutagenized oligonucleotide pools were purchased from Integrated DNA Technologies (Coralville, IA). These consisted of single-stranded DNA containing the lacUV5 promoter and LacI operator plus 20 bp on each end for PCR amplification and Gibson Assembly. Either both the lacUV5 promoter and LacI binding site or only the LacI binding site was mutated with a ten percent mutation rate per nucleotide. These oligonucleotides were amplified by PCR and inserted back into a pUA66-operator-GFP construct using Gibson Assembly. To achieve high transformation efficiency, reaction buffer components from the Gibson Assembly reaction were removed by drop dialysis for 90 minutes and cells were transformed by electroporation of freshly prepared electrocompetent cells. Following an initial outgrowth in SOC media, cells were diluted with 50 mL LB media and grown overnight under kanamycin selection. Transformation typically yielded 10⁶ − 10⁷ transformants as assessed by plating 100 μL of cells diluted 1:10⁴ onto an LB plate containing kanamycin and counting the resulting colonies.