Single stranded oligonucleotides

All chemicals and oligomers were used as received without any modification or further purification. Hexadecyltrimethylammonium bromide (CTAB, 99%), Cetyltrimethylammonium chloride solution (CTAC, 25 wt% in H₂O), Potassium Iodide (KI, 99.5%), L-ascorbic acid (AA, 99%) and tetrachloroauric(III) trihydrate (HAuCl₄·3H₂O, 99.9%) were purchased from Sigma-Aldrich. All single-stranded oligonucleotides were synthesized and purchased from Integrated DNA Technologies, Inc. (IDT). All aqueous solutions were prepared using high-purity deionized water (18.2 MΩ cm⁻¹).

ELPs and POPs: Single stranded oligonucleotides encoding the polymer genes were purchased from Integrated DNA Technologies (IDT) and cloned into a modified pet-24 vector via recursive directional ligation by plasmid reconstruction, as previously described, into chemically competent Eb5α E. coli to assemble the full-length polymer genes^{7 (link)}. In brief, A and B populations of each gene fragment were generated by restriction digest with AcuI and BglI and BseRI and BglI, respectively. Ligation of appropriate plasmid fragments from A and B populations following DNA gel purification resulted in the formation of a single, concatenated A + B gene fragment inside the modified pet-24 vector.
xPOPs: Following their full-length assembly using the above methods, xPOP genes were further isolated via BseRI and BamHI restriction digest, and the isolated gene was cloned into another modified vector with a pTac promoter and rrnB terminator instead of the T7 promoter and terminator of the original vector. The plasmids were then co-transformed into c321.ΔA E. coli alongside a pEvol tRNA/aaRS vector with two copies of pAcFRS.1.t1 synthetase. The C321.ΔA genome has previously been edited to remove all instances of the amber stop codon, and the tRNA/aaRS pair has been optimized to recognize the amber stop codon and incorporate para-azidophenylalanine^{47 (link),50 (link)}.

Single-stranded oligonucleotides used in this study (Supplementary Table S3) were obtained from Integrated DNA Technologies (Coralville, IA). ST2R24 library DNA used for the initial REPSA round was PCR amplified with primers ST2L and IRD7_ST2R for seven cycles to ensure maximal double-stranded DNA content with fully annealed randomized cassette regions. Subsequent REPSA round DNAs were PCR amplified for 6, 9, and 12 cycles to identify those products with optimal cassette integrity. Libraries for massively parallel semiconductor sequencing were prepared by a two-step fusion PCR process, using primers A_BC01_ST2R and trP1_ST2L as the initial set and A_uni and trP1_uni as the second set, as previously described [8 (link)]. Other duplex DNAs were prepared by conventional PCR amplification following the Taq DNA polymerase manufacturer’s instructions. EMSA probes were amplified with primers ST2L and IRD7_ST2R, while nucleic acids used in BLI assays were amplified with primers ST2L and Bio_ST2R. The concentrations for the modified oligonucleotides were measured with Qubit 3 Fluorometer following our protocol [37 (link)].