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. 2017 Aug 22;8:1597. doi: 10.3389/fmicb.2017.01597

FIGURE 3.

FIGURE 3

Determination of transcriptional antitermination of TRAM proteins. (A) E. coli RL211 carrying a cat gene cassette positioned downstream of the trpL terminator serves as the reporter. Termination (upper figure) and melting of the structured RNA terminator (below figure) allows expression of the chloramphenicol resistance gene cat. (B) Cells were transformed with pINIII vector alone or pINIII carrying cspE, Mpsy _0643, Mpsy_2002, Mpsy_3043, and Mpsy _3066, as indicated. Cultures were adjusted to an optical density at 600 nm of 1.0 by dilution with fresh medium and 10-fold serially diluted and spotted onto LB plates containing 100 μg/ml ampicillin with or without 0.2 mM IPTG (±IPTG) and 30 μg/ml chloramphenicol (±Cm) as indicated (Supplementary Figure S4B). Growths with Cm (100) and without Cm (10-3) were shown. (C) Using the antibody against TRAM2002, Western blot was performed to determine the abundance of the four TRAM proteins in E. coli RL211. The empty plasmid pINIII and bacterial CspE were included as negative controls. The amount of loaded total protein for each TRAM gene cloned strain is shown beneath the gel. ± on the top of each lane indicates the presence or absence of IPTG.