"PCR and Gibson assembly using cellular reagents. (a) Schematic depicting cellular PCR followed by cellular Gibson assembly for constructing new plasmids. Bacteria harboring target plasmids are mixed with polymerase-expressing cellular reagents and PCR is initiated by adding appropriate primers, buffer, and dNTP. The resulting PCR products are incubated with cellular reagents expressing Gibson assembly enzymes–Taq DNA polymerase, Taq DNA ligase, and T5 exonuclease–to assemble the new construct. (b) Cellular PCR amplification of vector and insert fragments directly from E . coli bacteria bearing target DNA plasmids using 2 x 10 7 cells of Phusion cellular reagents. Assembly parts include: (i) ""pATetO 6XHis full length"" vector for two part assembly with Kan r cassette bearing appropriate overlapping ends, and (ii) ""pUC19 Fragments 1 and 2"" for three part assembly with Kan r cassette whose ends overlap with pUC19 vector fragments. (c) Gibson assembly of agarose gel purified and unpurified cellular PCR products using pure or cellular Gibson assembly reagents. In ""negative control"" samples the PCR products were incubated in Gibson reaction buffer without pure or cellular Gibson enzymes. ""pATetO 6XHis + Kan r ""represents a two part Gibson assembly while ""Puc19 Fragment 1 + pUC19 Fragment 2 + Kan r "" represents a three-part Gibson assembly. Representative number of clones recovered in each case in the presence of both ampicillin and kanamycin are reported."