Constructing XPOPs via Recursive Ligation

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)}.