Characterization of <i>lac</i> operon regulation.

<p>A) Examples of the range of evolved LacZ activity phenotypes present in the four evolution environments. Degree of blue coloration on TGX plates gives a qualitative measure of LacZ activity. B) Schematic of <i>lac</i> operon and reporters used to measure <i>lac</i> operon regulation. <i>lacZ</i> encodes the β-galactosidase responsible for lactose catabolism and <i>lacY</i> encodes a lactose permease. The expression of <i>lacZYA</i> is directly controlled by LacI and CRP. LacI is a negative regulator, binding to operator sites within the <i>lacZYA</i> promoter (P_lac). LacI binding is inhibited by lactose and gratuitous inducers, such as TMG. CRP is a positive regulator, activating <i>lacZYA</i> expression when cAMP levels are elevated in response to low glucose concentrations. High levels of glucose also repress <i>lac</i> expression by inhibiting import of lactose through LacY. Two reporters were designed to measure LacI and CRP inputs into <i>lac</i> operon regulation. The native <i>lac</i> promoter drives expression of GFP and is subject to regulation by both LacI and CRP. A second reporter utilizes a mutant <i>lac</i> promoter that cannot bind LacI to drive expression of DsRedExpress2. This reporter is only subject to regulation by CRP. Solid lines indicate positive (arrows) and negative (blunt arrow) regulatory interactions; dotted lines indicate the transfer of metabolites; blue lines indicate the production of proteins; open arrows indicate expression start sites. Figure adapted from Ozbudak et al. 2004. C) Ancestral inducer response profile. Shown are flow cytometry histograms for the ancestor grown in a range of TMG concentrations. P_lac-GFP and P_lac(O-)-RFP measurements were taken simultaneously from the same cultures.