Quikchange method

To assess the ability of Hermes to carry out discrete steps of the transposition reaction, several assays were used. To assess cleavage, purified Hermes was incubated with plasmids containing either the full Hermes LE and RE (pHL2577 and variants) or the final 30 bp of each end (pRX1-Her). When mutated ends were used, changes were introduced using the QuikChange method (Agilent). After incubation, DNA was isolated and subjected to restriction digest to generate DNA fragments that could be identified after separation on an agarose gel. Cleavage assays were also carried out using oligonucleotides containing 60 bp of Hermes LE and 11 bp of flanking DNA.
To assess the ability of Hermes to generate hairpins when supplied with a pre- nicked LE, protein was incubated with a 71 bp oligonucleotide containing an intact bottom strand with a nicked top strand. For strand transfer, Hermes was incubated with oligonucleotides representing various lengths of pre-cleaved LE sequence and a pUC19 target plasmid.
For in vitro transposition into pGDV1, nuclear extracts of a Drosophila S2 cell line stably expressing Hermes were incubated with a Hermes donor plasmid and pGDV1. Recovered plasmid DNA was transformed into E. coli and the transposition events characterized.

Full-length DinF-BH and NorM-NG were expressed with cleavable hexa- and deca-histidine tags at their C-termini, respectively^{7 (link),9 (link)}. Mutations were introduced into the dinF-BH and norM-NG genes by using the QuikChange method (Agilent Technologies) and were confirmed by DNA sequencing^{7 (link),9 (link)}. Escherichia coli sBL21 (DE3) ΔacrABΔmacABΔyojHI cells^{34 (link)} were transformed with pET-15b vector containing the inserted genes encoding the DinF-BH and NorM-NG mutants.
The membrane expression levels of DinF-BH and NorM-NG were not affected by the mutations described in this study, as judged by western blot using an antibody against the His-tag (Supplementary Fig. 5). For the western blot, the antibody (Qiagen #34460) was diluted 2,500-fold before being mixed with the transfer membranes, and each sample examined on the gel was derived from cell membranes isolated from 80 µg E. coli BL21 (DE3) ΔacrABΔmacABΔyojHI cells expressing the MATE variants.

The MBP-fusion proteins used in this study were cloned from as reported before^{12 (link)} into the pET28 vector with an N-terminal MBP-His tag. The MtTfuA was cloned into the pETDuet-1 vector with an N-terminal His tag. The AzoTfuA and AzoYcaO were cloned into the first and second multiple cloning sites (MCSs) in pETDuet-1 vector, respectively, with an N-terminal His tag preceding the first MCS. These constructs were used for protein expression and as the templates for site-directed mutagenesis. Mutagenesis was performed using the QuikChange method (Agilent) with Q5 Polymerase (NEB). The primers used in this study were listed in Supplementary Table 1. All mutagenesis was verified by DNA sequencing.

The mCerulean fluorescent protein (FP) in a previous two-color FRET sensor^{17 (link)} was replaced with an mVenus FP using NheI and BglII restriction sites to create the homotransfer FRET GCK reporter. Point mutagenesis utilized the QuikChange method (Agilent) with the following primers; GCK(A456V), sense (5′-GGTCTCTGCGGTGGTCTGCAAGAAGGCTT-3′) and antisense (5′-AAGCCTTCTTGCAGACCACCGCAGAGACC-3′); GCK(K414E), sense (5′-CTCGGGTGCAGCTCGTACACGGAGCCA-3′) and antisense (5′-TGGCTCCGTGTACGAGCTGCACCCGAG-3′). The mVenus tandem dimer was constructed by removing GCK from the FRET GCK sensor using the BglII and BamHI restriction sites. The two mVenus FPs were ligated with a 10-amino acid linker sequence (SGLRSPPVAT) remaining between them. DNA sequencing (Genewiz, South Plainfield, NJ) was used to verify successful construction of all sensors.