Instructions for Imaging PROPS in E

7/15/2011

Cohen Lab

Harvard University

617-496-9466

Instructions for imaging PROPS in E. coli[1]

The proteorhodopsin optical proton sensor (PROPS)[2] provides a fluorescent readout of membrane potential in bacteria. It is also sensitive to cytoplasmic pH, showing brighter fluorescence at lower pH.

PROPS is very dim, roughly 1,000 times dimmer than GFP. Imaging on a conventional cell biology epifluorescence microscope is not likely to work. Our microscope is adapted from a system designed for single-molecule fluorescence. The key ingredients are:

1) Inverted microscope. We have a homemade microscope, but a commercial inverted microscope should work too. For single-cell work we often image in through-the-objective TIRF or "pseudo-TIRF", i.e. glancing angle of incidence. Doing so decreases the background from autofluorescence of the imaging medium.

2) A fairly intense laser configured for wide-field illumination. A 10 mW 632 nm HeNe is adequate, though a 100 mW 635 diode is preferable. We use a 100 mW 638 nm source from CrystaLaser, part # DL638-100-O.

3) A high NA objective. We use either a NA 1.2, 60x water immersion objective or a NA 1.45, 60x oil immersion objective.

4) A good filter set. A Cy5 filter set appropriate for single-molecule imaging will work. The emission filter should pass approximately from 650 - 750 nm.

5) A highly sensitive camera. We use an iXon+ 897 EMCCD from Andor. Other manfucaturers' cameras with the same e2v CCD 97 image sensor should work too.

Cell culture

Grow E. coli to early-log phase (OD600 = 0.3 – 0.4) in 50 mL of LB medium in a shaking incubator at 33 C. Add inducer along with all-trans retinal (5 mM final concentration from a 20 mM stock in ethanol) and conduct further growth in the dark. Harvest cells after 3.5 hours by centrifugation (5000 RPM for 5 minutes) and wash with 30 mL of minimal medium (1x M9 salts, 0.4% glucose, pH 7). Centrifuge cells again and resuspend cells in 5 mL minimal medium and use immediately or store at 4 C for later use.

The JK1 plasmid contains PROPS in a pBAD TOPO backbone, with Arabinose as the inducer and Ampicillin as the selection antibiotic.

PROPS does not localize to the plasma membrane in eukaryotic cells. We are developing voltage indicators that function in eukaryotes. Stay tuned...

[1] Updated information on applying and imaging microbial rhodopsin voltage sensors can be found at: https://www2.lsdiv.harvard.edu/labs/cohen/Resources/Resources.htm

[2] J. Kralj, D. R. Hochbaum, A. D. Douglass, A. E. Cohen, “Electrical spiking in Escherichia coli probed with a fluorescent voltage-indicating protein,” Science, 333, 345-348 (2011)