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. Author manuscript; available in PMC: 2009 Apr 8.
Published in final edited form as: Biotechnol Bioeng. 2009 Feb 1;102(2):357–367. doi: 10.1002/bit.22097

Figure 1.

Figure 1

A: Design of the expression vectors pASKCB1 and pASKCB1-GFP. Ptet: tetracycline promoter; RBS: ribosome-binding site; FLAG: FLAG octapeptide epitope; CB1: E. coli codon-optimized gene encoding full-length human central cannabinoid receptor; His8: octahistidine tag; TEV: recognition sequence for the tobacco etch virus protease; GFP: green fluorescent protein variant GFPmut2 optimized via FACS. B: Schematic representation of the screening process. E. coli MC4100A cells were subjected to transposon mutagneesis, and FACS was employed to screen the generated MC4100A::Tn5 (pASKCB1-GFP) library and isolate E. coli transposon insertion mutations that confer higher fluorescence due to an increase in the amount of membrane-bound CB1-GFP. For FACS screening, cells were initially gated based on size (gate P1) on a side-scatter (SSC-H) versus forward-scatter (FSC-H) plot. Subsequently, the clones corresponding to the top 1-3% fluorescent events (gate P2) were isolated and subjected to repeated rounds of FACS sorting.