Fig. 2. Continuous evolution of TnsABC.

(A) Summary of TnsABC evolution campaign. Whether evolution segments were conducted using PANCE or PACE is specified, with PANCE passages or PACE hours indicated. Circuit architectures are shown in fig. S2, A to C. (B) Overnight phage propagation assays with wild-type (WT) TnsABC SP, pooled evolved SPs from each evolution segment, and gIII-expressing phage (positive control for propagation). X-axes indicate host E. coli variants encoding circuit 1.0. Host A was used for PANCE N1. Hosts B and C are of increased selection stringency, manipulated by reducing the promoter strength in the transposon on CP2 (Hosts B and C) and reducing the ribosome binding site upstream of gIII on the AP (Host C). Host NT A is host A with a non-targeting crRNA. The left graph shows phage propagation levels (output phage titer divided by input titer). The right graph shows transposon integration efficiencies at the AP target site in E. coli following overnight propagation, as measured by qPCR. (C) Genotypes of a subset of evolved TnsABC variants. Variants N1–1, P1–3, and N2–1 showed the highest integration activity among the variants emerging from their respective PANCE or PACE experiments at two tested genomic sites in HEK293T cells (fig. S6). Variants P2–2, P2–7, and P2–11 are representative of the genotypes that emerged from P2. (D) 1-kb transposon integration efficiencies at two genomic loci in HEK293T cells for wild-type (WT) and evolved TnsABC variants specified in (C). (E and F) Assessing the contributions of evolved TnsAB and TnsC subunits to overnight phage propagation levels on P2 host E. coli (E) and 1-kb transposon integration efficiency in HEK293T cells (F) for representative P2 CAST variants. Data in (B) and (D–F) are shown as mean±s.d. for n=3 independent biological replicates.