Construction of the TransBac empty (pTB-empty) plasmid
The single-copy TransBac library plasmid coding ybeX was purified
using an in-house alkaline lysis method followed by purification via
FavorPrep™ plasmid DNA extraction mini kit (Favorgen, #FAPDE300). The
cloning site was sequenced by Sanger sequencing (University of Tartu).
The ybeX coding region was removed via restriction enzyme
cleavage of FastDigest XmaJI and Sfi I (Thermo
Scientific). The sticky ends were filled using Klenow fragment (Thermo
Scientific), and the linear plasmid was ligated using T4 DNA ligase
(Thermo Scientific™) following manufacturer protocols. The ligation
reaction was transformed into Inoue E. coli DH5α chemical
competent cells (Green and Sambrook, 2020), and the TransBac empty
backbone plasmid was purified as mentioned above. The size of the
plasmid DNA was determined via agarose gel electrophoresis, and the
cloning site was sequenced using SO10 primer (see Table S3). The plasmid
was electroporated into wild-type and ΔybeX strains.