Construction of pBJ vectors

The pBBR1-MCS5 derivative pJN105 was cut with XhoI and NotI. The larger fragment was treated with T4 DNA polymerase followed by circularization with T4 DNA ligase, yielding pJN105ΔpBAD. Two DNA fragments encoding tnsABCD or luxCDABE were amplified by PCR from pBEN276 (Howe et al., 2010) using the primer sets Pfrr_inside_F plus New_Tn7L_pJN_M13F or Pfrr_inside_R plus pBEN_Pamp_pJN_M13R, respectively. These DNA fragments were assembled using the NEBuilder HiFi DNA Assembly Cloning Kit (NEB, E2621) into ApaI-digested pJN105ΔpBAD, yielding pBJ1. The kanamycin promoter (P_kan) was amplified from pBBR1-MCS2 using Pkan_pBJ_F2 and Pkan_pBJ_R2 and assembled into XhoI-digested pBJ1 to replace the frr promoter (P_frr), resulting in pBJ2.

pBJ3 and pBJ4 were designed to confer kanamycin resistance instead of the gentamicin resistance conferred by pBJ1/2. A DNA fragment was amplified from pBEN276 using Pfrr_inside_R plus pBEN_Pamp_pJN_M13R, and another DNA fragment was amplified from pBJ1 using Pfrr_inside_F plus pBJ_dXhoI_Tn7R. These two fragments were then assembled into ApaI-digested pBBR1-MCS2, yielding pBJ3. pBJ4 was constructed by replacing P_frr in pBJ3 with P_kan as described above.

To broaden the host range of the pBJ vectors, a broad-host-range RK2 replicon was amplified from pFREE-RK2 using OriV_RK2_Fwd plus OriV_RK2_Rev and assembled into NaeI-digested pBJ1 and pBJ2 to produce pBJ5 and pBJ6, respectively. For a similar purpose, the broad-host-range cosmid vector pLAFR3 was cut with EcoRI and treated with T4 DNA polymerase, followed by further digestion with PstI. This linearized vector was used as a recipient for a NaeI-SbfI restriction fragment of pBJ2 containing the tnsABCD-Pkan-luxCDABE cassette, yielding pBJ7.

Primers used for the construction of pBJ vectors are listed in Supplemental Table 2.