Materials

BL21-Gold(DE3) Cells (Stratagene, La Jolla, CA, USA)

Glycerol (Merck, Nottingham, UK)

IPTG (Sigma, Madrid, Spain)

Arabinose (Sigma)

Nikon Eclipse-600 Microscope equipped with a 100× oil immersion objective, a filter set for GFP (Nikon, Badhoevedorp, The Netherlands)

Nikon DS-5Mc Cooled Camera (Nikon)

Complete Protease Inhibitor Cocktail (Roche, Barcelona, Spain)

Lysozyme (Sigma)

Bio-Rad IQ5 System (Bio-Rad Laboratories, Madrid, Spain)

β-Mercaptoethanol (Sigma)

DDT (Sigma)

2× Laemmli Sample Buffer (Invitrogen, Barcelona, Spain)

Ni-Sepharose 6 Fast Flow (Amersham Biosciences, Barcelona, Spain) Vector Plasmids

Expression vectors encoding the N- and C-terminal segments of the GFP mutant sg100 fused to two leucine zipper-coding sequences (designed as NGFP-LZ1 and LZ2-CGFP) and two control vectors (designated as CGFP and NGFP without LZ1 or LZ2 sequences) were a generous gift of Thomas J. Magliery (1, 2). The sequence files containing detailed vector descriptions are available on the Regan Laboratory web site (www.yale.edu/reganlab/publications.html). Bacterial Transformation

BL21-Gold(DE3) cells were simultaneously transformed with the appropriate plasmid pairs in accordance with the manufacturer's recommendation. Doubly transformed cells (NGFP-LZ1, LZ2-CGFP) were plated on doubly selective LB agar plates (100 µg/ml ampicillin and 35 µg/ml kanamycin) and incubated at 37°C overnight. Individual colonies were picked up, each resuspended in 10 ml of selective LB media, incubated at 37°C for 8–10 h, and kept in 20% glycerol at -80°C until use. As negative controls, matched cells that were cotransformed with CGFP + NGFP, NGFP-LZ1 + CGFP, and NGFP + LZ2-CGFP were included in each experimental setting. Microscopic Detection of GFP Reassembly in Bacterial Cells

A fresh liquid cell culture taken in exponential growth phase (at an optical density of 0.5 at 600 nm) was induced in a presence of 40 µM IPTG and 0.2% arabinose and allowed to continue growth at 27°C for an additional 8–10 h. 5–10 µl of cell suspension was removed each hour to detect the cells that exhibit fluorescence (thus having properly reassembled GFP) under a Nikon Eclipse-600 microscope equipped with a 100× oil immersion objective, a filter set for GFP, and a Nikon DS-5Mc cooled camera. We routinely examined at least 1000 cells per sample, and each experiment was replicated at least three times. Green fluorescence typically developed after 1–4 h of incubation. Spectrofluorometric Detection of GFP Reassembly in Cell Lysates

Cotransformed cells from liquid cultures were harvested by centrifugation (5,000× g, 20 min, 4°C) at various time periods after induction and resuspended (1:10 v/v) in TBS-T buffer (50 mM Tris-HCl, pH 8.0, 400 mM NaCl, 0.2% Triton X-100) supplemented with complete protease inhibitor cocktail. The cells were lysed using 2 µg/µl lysozyme at 4°C for 30 min followed by DNase treatment (at 0.05 µg/µl) at 20°C for 20-30 min. Resulting cell lysates were centrifuged (20,000× g, 30 min, 4°C), and the supernatants were used in downstream spectrofluorometric or purification assays. The spectrofluorometric assay was started by adding cell lysates (from 5 to 100 µl) to 0.2 ml PCR tubes with optical caps, and readings were collected using a Bio-Rad IQ5 system. In the whole plate mode, the optical detection module captures a common image on which reassembled GFP samples appear as white spots over the black background. The data analysis module generates the values [in relative fluorescent units (RFUs)] corresponding to the intensity of selected positive green fluorescence signals. The values were corrected by subtracting the background signal of appropriated negative controls. Cell Solubilization for SDS-PAGE Analysis and Protein Purification

Cell pellets were resuspended (1:10 v/v) in 2× Laemmli sample buffer supplemented with 10% β-mercaptoethanol, 100 mM DDT, and complete protease inhibitor cocktail and boiled at 100°C for 5 min. After centrifugation at 20,000× g for 30 min at 15°C, the supernatants were analyzed by SDS-PAGE in 15% Tris-Glycine gels, followed by staining with Coomassie Blue. For His-6 affinity purification, TBS-T supernatants prepared as just described were loaded at 4°C on Ni-Sepharose 6 Fast Flow columns for at least 1 h. After washing with 20 bed volumes of TBS-T buffer containing 30 mM imidazole, the proteins were eluted in TBS buffer containing 500 mM imidzole and analyzed by SDS-PAGE. Correspondence

Address correspondence to Mario Torrado, Institute of Health Sciences, University of La Coruña, Campus de Oza, Building “El Fortín,” La Coruña, 15006, Spain. e-mail: [email protected]