Part:BBa_K773002:Experience

Characterization

Proteorhodopsin

We successfully constructed the part for proteorhodopsin, using PCA assembly based on the information from the paper Light-powering Escherichia coli with proteorhodopsin, as can be verified by our sequencing data in the parts registry. We then conducted an ATP assay on our proteorhodopsin strain to determine if it effectively produced a proton gradient to generate more ATP. Unfortunately our results were inconclusive. In this experiment, we expected to see increased ATP generation when the promoter was induced and light was available for the proteorhodopsin pump. We used cyanide (CN) to inhibit the electron transport chain, and covered tubes in foil to imitate darkness. A 60 Watt incandescent light bulb was used for excitation. aTc was used to bind to the R0040's tetR repressor. Luminescence correlates to ATP concentration.

In the J123106 strain, we saw an unexpected slight increase in luminescence when there was no light source, meaning the proteorhodopsin did not yield increased ATP. The cells treated with CN did not produce markedly higher levels of ATP, suggesting that a 1 mM concentration of cyanide may have been insufficient in shutting down the electron transport chain.

Similarly in the R0040 strain, when aTc was bound to the promoter repressor, we saw only a slight increase in luminescence corresponding to ATP yield. When the repressor was not bound (no aTc was present) the levels of ATP were about the same as when the repressor was bound, signifying the promoter may be a little "leaky".

When we sequenced the proteorhodopsin strain, we found that we had the sequence we expected. However, there are many reasons that our proteorhodopsin did not behave as we expected in the characterization assay. Missing from our part is the ribosome binding site; this could cause lack of expression of the gene. Another issue may be our protocol for reading luminescence. We plan to add a ribosome binding site and research our plate reader protocol for further characterization of the part.

User Reviews

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••••• 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.