Kpni and hindiii

The three zebrafish TG2 coding sequences have been cloned in the pET30a(+) bacterial expression vector in order to produce the three TGs2 as His6-tagged recombinant proteins. Zebrafish TGs sequences were PCR-amplified with Phusion™ High-Fidelity DNA Polymerase (Thermo Scientific, Waltham, MA USA, cat#F530S), using 2 dpf zebrafish embryos cDNA as template and zTGs2-specific primers, designed to insert restriction sites for KpnI and HindIII, respectively, at the 5′ and 3′ of the coding sequences (Table 2). The PCR products were gel purified with the GenElute™ Gel Extraction Kit (Sigma-Aldrich, Burlington, MA, USA, cat#NA1010), following the manufacturer’s instructions, and digested with KpnI and HindIII (New England BioLabs, Ipswich, MA, USA, cat#R3142 and cat#R3104) together with the recipient plasmid. The digested vector and DNA fragments were ligated with T4 DNA ligase (Thermo Scientific, cat#EL0011) and cloned into DH5α competent cells. Plasmid minipreps were obtained from overnight culture at 37 °C in 2xTY medium with GenElute™ Plasmid Miniprep Kit (Sigma-Aldrich, cat#PLN70), and transformed into BL21(DE3) competent cells.

K. pneumoniae MKP103 genomic DNA was extracted using Easy-DNA Kit (Invitrogen). PCR amplification was performed with primers KpyqjA1 and KpyqjA2 for cloning Klebsiella YqjA and primers KpyghB1 and KpyghB2 for cloning Klebsiella YghB (Table S1). The DNA fragments were purified and digested with KpnI and HindIII (New England Biolabs). pBAD Apr^r plasmid was similarly digested and dephosphorylated. A 20-µL ligation reaction using Hi-T4 DNA ligase (New England Biolabs) was incubated at 25°C for 3 hours, then transformed into SM10 (λ pir) (86 (link)) competent cells. Positive transformants were selected on 50 µg/mL Apr.