iGEM15_UNITN-Trento

This part was previously found by other users to not express protein nor produce ATP. We improved this part by adding a strong RBS and using a different inducible promoter (arabinose, araC-pBAD). We were able to express the protein and functionally characterize it. The part now works! To see our complete results check out our part BBa_K1604010 and our Wiki UNITN-Trento iGEM 2015! Part improved by Silvia Galvan (iGEM Trento 2015)

Figure 1. Proteorhodopsin needs retinal to be expressed properly. Panel on the left NEB10β cells transformed with BBa_K1604010 and grown in LB and induced in LB or M9 Minimal Media with 5 mM arabinose and 10 μM of retinal at 30 °C or 37 °C. Negative controls were cells transformed with BBa_K731201 (i.e. araC-pBAD). By the screening of several parameters (media, temperature, time of induction) we discovered that the optimal expression conditions were in LB, at 37 °C overnight in the presence of 10 μM of all-trans retinal. Panel on the right Proteorhodopsin is successfully expressed in M9. Cells transformed with BBa_K1604010 and BBa_K731201 were grown in LB and transferred in M9 at an OD of 0.6 and induced with arabinose with the presence of 10 μM of retinal. After 6 hours of induction the cells were centrifuged and the supernatant was discarded. From left to right: araC-pBAD induced with retinal (A), proteorhodopsin induced with retinal (B), proteorhodopsin induced (C) and not induced (D)both without retinal. Although LB gives the maximum expression as shown in the SDS-Page, we were able to successfully express Proteorhodopsin also in M9.

Figure 2. More ATP under anaerobic condition! E. coli NEB10β transformed with BBa_K1604010 (Proteorhodopsin) and BBa_K731201 (araC-pBAD) were grown in LB at 37 °C until an OD of 0.6 and induced in LB with 5 mM arabinose and 10 μM retinal in the dark. After 5 hours of induction at 37 °C the culture were transferred in sealed bottles in the anaerobic chamber. Sample in the dark (in purple) were kept in aluminum foil. Light-exposed samples (in yellow) were excited with a 160 W halogen light bulb placed outside the incubator. After an overnight incubation an ATP assay was performed. The commercial kit used measures ADP/ATP ratio. Proteorhodopsin-engineered E. coli exposed to light and under anaerobic conditions show a much lower ADP/ATP ratio (i.e. high ATP) in comparison to control cells (araC-pBAD and dark condition), meaning that the cells are growing. The test confirms that proteorhodpsin supports E. coli viability under anaerobic condition when cells are light-exposed. The proton pump showed some activity in the dark.