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, Austria). The
cloning site was sequenced by Sanger sequencing. The ybeX coding
region was removed via restriction enzyme cleavage of XmaJI andSfiI (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. The plasmid was electroporated into
Keio collection strains BW25113 and ∆ybeX .