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1.
Fig 1

Fig 1. From: Construction of a Broad-Host-Range Tn7-Based Vector for Single-Copy PBAD-Controlled Gene Expression in Gram-Negative Bacteria.

A genetic map of the features of the PBAD-based mini-Tn7T-Tp-araC-PBAD-MCS delivery vector, pTJ1. (A) The map of pTJ1 was assembled using Gene Construction Kit version 3.04 (Textco). Abbreviations: araC, gene encoding the repressor of PBAD; bla, β-lactamase-encoding gene; dhfRII, gene encoding trimethoprim-resistant dihydrofolate reductase; FRT, Flp recombinase target; MCS, multiple-cloning site (see panel B for restriction enzyme cleavage sites); oriT, origin of transfer; PBAD, arabinose-inducible promoter; RBS, ribosome-binding site; Tn7L and Tn7R, Tn7 left and right ends, respectively. (B) Sequence encompassing PBAD and multiple cloning site of pTJ1. Bold letters indicate the −10 and −35 sequences of the PBAD promoter and a consensus RBS. Unique restriction enzyme cleavage sites suitable for cloning of exogenous DNA fragments are shown.

F. Heath Damron, et al. Appl Environ Microbiol. 2013 January;79(2):718-721.
2.
Fig 2

Fig 2. From: Construction of a Broad-Host-Range Tn7-Based Vector for Single-Copy PBAD-Controlled Gene Expression in Gram-Negative Bacteria.

Western blot analysis of IMPDH-HA expressed in P. aeruginosa. (A) Detection of IMPDH-HA expressed from the PBAD promoter of mini-Tn7T-Tp-araC-PBAD-MCS integrated into the P. aeruginosa strain PA14 chromosome. The pTJ1-guaB-HA expression construct was integrated into the chromosome. Western blot analysis using anti-HA high-affinity antibody (clone 3F10; Roche Applied Science) (16) was used to detect the HA-tagged IMPDH in minimal media lacking (−) or containing (+) 1% (wt/vol) arabinose. The contents of the lanes are as follows: lane 1, P. aeruginosa strain PA14 without HA-epitope-tagged guaB as a control for antibody cross-reactivity; lanes 2 and 3, PA14 mini-Tn7-guaB-HA; lane 4, PA14 pHERD20T; lanes 5 and 6, PA14 pHERD20T-guaB-HA. IMPDH-HA was detected when guaB-HA was expressed from the PBAD promoter from either the autonomously replicating pHERD20T plasmid (lane 6) or following chromosome-encoded guaB-HA from pTJ1-guaB-HA (lane 3). Samples in lanes 4 to 6 were grown in the presence of 300 μg/ml carbenicillin for maintenance of pHERD20T. (B) Detection of IMPDH-HA expressed from the PBAD promoter from strains grown in minimal media in the presence of different concentrations of arabinose. Western blot analysis was performed as previously described. All lanes contained PA14 mini-Tn7-guaB-HA. The concentrations of arabinose used were (wt/vol) 0.01%, 0.10%, 0.50%, and 1.00% (lanes 1 to 4, respectively).

F. Heath Damron, et al. Appl Environ Microbiol. 2013 January;79(2):718-721.

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