Figure 2.

Tn3.1^MIN reduces levels of the TEM-1 β-lactamase. (A) Illustration of the pET15b-sfgfp expression plasmid, which contains the Tn3.1 fragment. (B) BL21(DE3) harboring the pET15b-sfgfp (Tn3.1) expression plasmid was plated on LB agar containing different concentrations of ampicillin or carbenicillin. Colony numbers were normalized by the number of colonies that grew in the absence of antibiotics. The minimum inhibitory concentration (MIC₉₀) required to kill 90% of cells was extrapolated from the curve (dotted line) and deemed to be approximately 700 μg/mL for ampicillin and >5000 μg/mL for carbenicillin. (C) BL21(DE3) cells (without an expression plasmid) were plated on LB agar containing no antibiotic or 1 μg/mL ampicillin or carbenicillin. As growth was not observed on 1 μg/mL ampicillin or carbenicillin, the MIC₉₀ was deemed to be <1 μg/mL. (D) BL21(DE3) harboring the pET15b-sfgfp expression plasmids plated on LB agar containing different concentrations of ampicillin. The pET15b-sfgfp expression plasmids, denoted C2, B6, D3, and C5, were selected from a directed evolution process and contained Tn3.1 fragments with a different translation initiation region (TIR) for bla. C2 was chosen for further characterization and was named Tn3.1^MIN. (E) Nucleotide sequence alignment of the TIR for bla in Tn3.1, Tn3.1^MIN (C2), B6, D3, and C5. The TIR is defined as the nucleotide sequence from the Shine–Dalgarno (SD) region through to the fifth codon.¹⁸ (F) As in panel (B) except that BL21(DE3) harbored the pET15b-sfgfp (Tn3.1^MIN) plasmid. The MIC₉₀ was deemed to be <30 μg/mL for ampicillin and <200 μg/mL for carbenicillin. (G) Levels of TEM-1 β-lactamase in BL21(DE3) harboring the pET15b-sfgfp expression plasmid (Tn3.1 or Tn3.1^MIN), or the culture media, were probed by Western blotting with antisera to the TEM-1 β-lactamase.