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. Author manuscript; available in PMC: 2013 Jan 10.
Published in final edited form as: Gene. 2011 Oct 13;491(2):116–122. doi: 10.1016/j.gene.2011.10.013

Figure 3.

Figure 3

Inhibition of CNF1 expression by truncated GidA is mRNA context dependent.

A. Schematic representation of different regions of cnf1 gene fused with β-lactamase reporter gene. AA indicates the amino acid regions in each construct that were being fused to Bla. For constructing each plasmid, the primers used were listed; their detailed sequence information is shown in table 2. For each reaction, 2 μg bacterial lysates were incubated with 0.1 mM nitrocefin, after 30 minutes of incubation; 1 mM tazobactam sodium was added to stop the reaction. The numbers in column RS218 and NBC-28G9 represent the average and standard deviation of three measurements of OD486 (Bla activity, means ± standard deviations) of each construct in strain RS218 and NBC-28G9, respectively. In NBC-28G9, CNF1-Bla that was expressed from its genome has been determined by strain NBC-28G9 without plasmid constructs. It was extremely low in the diluted bacterial lysates when tested at the same condition (The average OD486 reading was 0.016). The statistical significance of the differences in Bla activity between two strains with the same plasmid construct was calculated with t test (*, p<0.05; **, p<0.01). The constructs without * label in p column indicate that the CNF1 fragment and Bla fusion proteins from those constructs were produced at similar level between RS218 and NBC-28G9.

B. Western blot analysis of the expression of CNF1-Bla fusion proteins from artificial promoter trc in plasmid in strains RS218 and NBC-28G9. 50 μg bacterial crude extracts were loaded in each lane and then separated with 4–12% Bis-tris gel (Invitrogen). β-lactamase polyclonal antibody (Millipore) was used as the primary antibody. Different constructs in different strains were indicated as labeled. “*” denotes non-specific band. The intensity of non-specific band on the film indicates that the same amount of protein was loaded in each well. The position and size of CNF1-Bla expressed from pCXN, pCXN118 and pCXN128 is indicated along with the arrow.

C. Reverse transcribed PCR analysis of cnf1 mRNA in RS218 and NBC-28G9. House-keeping gene rplU was used as the marker for the integrity of mRNA and the loading control. “−” denotes negative control without reverse transcriptase. “+” denotes reactions with reverse transcriptase.