The function of the predicted CRP-binding site of Shewanella lipBA operon was assessed in vitro using electrophoretic mobility shift assays (EMSA) with little improvements (Feng and Cronan 2011a (link); Goble et al. 2013 (link); Feng et al. 2014 (link)). In the EMSA tests, nine pieces of DNA probes were composed of seven suspected probes (lipBA_she, ybeD_ec, ybeD_es, ybeD_kp, ybeD_st1, ybeD_st2, and ybeD_yp) and the two control probes, the fadD_ec site with known function (the positive control) and the lipA_ec without any function (the negative control) (Table3 ). The digoxigenin (DIG)-labeled DNA probes were prepared in vitro through annealing two complementary oligonucleotides in TEN buffer (10 mmol/L Tris-HCl, 1 mmol/L EDTA, 100 mmol/L NaCl; pH 8.0) and then labeled by the terminal transferase with DIG-ddUTP (Roche, Indianapolis, IN, USA) (Feng et al. 2014 (link)).
In the presence/absence of cAMP (20 pmol), the various DIG-labeled DNA probes (0.2 pmol) were incubated with or without CRP protein in the binding buffer (Roche) at room temperature for around 20 min. Following the separation of the DNA-protein complexes with a native 7% PAGE gel, the chemiluminescent signals were further captured by the exposure to ECL film (GE Healthcare, Piscataway, NJ, USA) (Feng and Cronan 2011b (link), 2012 (link)).
In the presence/absence of cAMP (20 pmol), the various DIG-labeled DNA probes (0.2 pmol) were incubated with or without CRP protein in the binding buffer (Roche) at room temperature for around 20 min. Following the separation of the DNA-protein complexes with a native 7% PAGE gel, the chemiluminescent signals were further captured by the exposure to ECL film (GE Healthcare, Piscataway, NJ, USA) (Feng and Cronan 2011b (link), 2012 (link)).
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