Construction of deletion library
pGFPUV vector (Clontech, Mountain View, CA) was used as the transposon target plasmid for in vitro transposition reaction. GFPUV protein carrying a 24 amino acid N-terminal tag is constitutively expressed from this vector. All of the four Mly I sites (249, 2334, 2836 and 3322) in pGFPUV vector were removed by site-directed mutagenesis without changing the amino acid sequence (see Additional file 4: Table S2 for primers). The engineered Mu transposon bearing chloramphenicol-resistant gene and Mly I sites was constructed as described . Transposon DNA was released from pUC19 vector by Bgl II digestion, gel purified and then resolved on a 1 % agarose gel to determine DNA concentration and purity. Transposition reaction was performed in a 20 μL mixture containing 50 mM Tris-acetate, pH 7.5, 150 mM potassium acetate, 10 mM magnesium acetate, 4 mM spermidine, 570 ng of pGFPuv vector, 140 ng of transposon DNA (~1.3 molar excess) and 1 unit of HyperMu MuA transposase (Epicentre Biotechnologies, Madison, WI). The reaction was kept at 30 °C for 4 hrs and stopped by addition of 0.1 % SDS, followed by heat-inactivation at 70 °C for 10 min. The reaction product was transformed into chemically competent GeneHogs Escherichia coli cells and plated on LB agar containing 100 μg/mL ampicillin and 10 μg/mL chloramphenicol for selection of pGFPUV vector with transposon insertion. Approximately 38,000 colonies were collected and maxi-prepped to build the pGFPUV-MuDel library.
pGFPUV-transposon library DNA was digested with EcoR I/Kpn I, and the 2.0 kb DNA fragment corresponding to transposon-carrying GFPUV gene was re-ligated with the 2.6 kb vector backbone. This purified pGFPUV-transposon library was then digested with Mly I to remove transposon DNA, leaving a three nucleotide scar at a random position of GFPUV. The blunt-end intramolecular ligation was performed in a 20 μL reaction containing 50 mM Tris–HCl, pH7.5, 10 mM MgCl2, 10 mM dithiothreitol (DTT) and 0.5 mM ATP, 300 ng DNA, 400 cohesive end units of T4 DNA ligase (NEB). The ligation product was transformed into chemically competent GeneHogs E. coli and the transformants were plated on LB agar containing 100 μg/mL ampicillin. In total, 10,000 colonies were collected, and maxi-prepped to build the triplet nucleotide deletion library.
Screening for deletion mutants
The deletion library DNA was transformed into GeneHogs and the transformants were plated on LB agar supplemented with 100 μg/mL ampicillin at a density of 500 colonies per plate. Transformed cells were grown at 20 °C for 30 h and screened for fluorescence by visual inspection using Spectronics (Westbury, New York) model TC312E UV transilluminator under 310 nm wavelength. Forty fluorescent and 24 non-fluorescent colonies were selected and sequenced for further characterization.
Liquid culture whole-cell fluorescence
Plasmids encoding wild-type GFPUV (wtGFPUV) and deletion mutants were transformed into GeneHogs and plated on LB agar containing 100 μg/mL ampicillin at a density of ~200 colonies per plate. Single colonies with a diameter of ~0.5 mm were inoculated into 2 mL of liquid LB medium supplemented with 100 μg/mL ampicillin and grown at 37, 30 and 23 °C for 14, 18 and 25 h, respectively. Cells were harvested by centrifugation, washed with 500 μL TNG buffer (100 mM Tris–HCl, pH 7.5, 150 mM NaCl and 10 % glycerol) and then resuspended in 100 μL TNG buffer . One milliliter of cell resuspension with an OD600 of 0.150 ± 0.003 was prepared for fluorescence assay and the remaining cells in TNG buffer were stored at −20 °C for further experiments. Whole-cell fluorescence was determined using a model F4500 fluorescence spectrophotometer (Hitachi, Tokyo, Japan) as described . The excitation and emission wavelength was set as 397 nm and 509 nm, respectively. Background fluorescence of empty GeneHogs cells was subtracted from each reading. All data were normalized to those of wtGFPUV under the same conditions. Three experimental replicates were performed for each sample.
Protein expression and fraction soluble
For wild-type and mutant GFPUV, 300 μL cell suspension in TNG buffer with an OD600 of 0.100 was sonicated by Branson model 450 sonicator (Branson Ultrasonics, Danbury, CT) equipped with a 1/2 inch horn and a 1/8 in. tip. The power output and duty time were both set 50 %. Cell suspension was forced to two sequences of 10 pulse sonication with an interval of 3 min. Following sonication, 150 μL cell lysis was centrifuged at 12,000 g for 10 min and the supernatant was transferred into a new tube. SDS loading samples were prepared in a 30 μL solution containing 15 μL crude cell lysis (whole protein) or supernatant (soluble fraction) and 15 μg bovine serum albumin (BSA) protein (Sigma, St. Louis, MO) as an internal standard. Protein samples were resolved in 12 % acrylamide SDS-PAGE gels and analyzed by Image J (http://rsbweb.nih.gov/ij/). GFP expression was quantified based on the density ratio of GFP and BSA bands as described . The fraction soluble of each sample was extrapolated from the density ratio of soluble GFP present in supernatant and overall GFP present in crude cell lysis. Three experimental replicates were performed for each sample.
Protein expression and purification and determination
wtGFPUV and mutants with internal deletions were sub-cloned into pET28b(+) vector (EMD Chemicals Inc., San Diego, CA) using primers CTAgctagcATGAGTAAAGGAGAAGAACTT (Nhe I site in lowercase) and CCCaagcttTTATTTGTAGAGCTCATC (Hind III site in lowercase). pET28b vector encoding wild-type and mutant GFPUV with N-terminal His6 tags were transformed into E. coli BL21 (DE3) cells (Stratagene Inc., La Jolla, CA) and spread on LB agar plates supplemented with 50 μg/mL kanamycin. A single colony was inoculated into 500 mL LB media supplemented with 50 μg/mL kanamycin and grown at 37 °C to an OD600 of 0.8. Protein expression was induced with 1 mM isopropyl-β-D-1-galactopyronaside (IPTG) for 12 h at 20 °C. The next day, cells were centrifuged and re-suspended in 25 mL binding buffer (100 mM HEPES, pH 7.5, 5 mM imidazole, 1 mM phenylmethanesulfonyl fluoride, PMSF). Cells were lysed by sonication and then centrifuged at 12,000 g for 30 min. Supernatant was transferred into a new tube and then loaded on to a column pre-packed with 1 mL HisLink resins (Promega Corporation, Madison, WI). The protein-bound resins were washed with 20 mL wash buffer (100 mM HEPES, pH 7.5, 20 mM imidazole) and then eluted with 5 mL elution buffer (100 mM HEPES, pH 7.5, 300 mM imidazole). Purified proteins were concentrated and buffer-exchanged into TNG buffer (100 mM Tris–HCl, pH 7.5, 150 mM NaCl and 10 % glycerol). The concentration of purified proteins were determined using Pierce BCA Protein Assay kit (Thermo Fisher Scientific, Rockford, IL).
Biophysical properties of deletion mutants
The excitation and emission scan were performed using 6 μg/mL purified proteins in TNG buffer. An emission wavelength of 509 nm was used for excitation scan, while emission scan was performed using excitation wavelengths of 397 nm and 495 nm.
The concentration of matured GFP was determined using “base-denatured” method . Briefly, 7 μM purified proteins were denatured in 0.1 M NaOH for 5 min at 25 °C. Absorption spectra from 300 nm to 600 nm were recorded. The concentration of matured GFPs was calculated from experimentally determined A447 and previously reported ε447 (44,000 M−1 cm−1 for denatured GFPs)  using Beer’s Law. The efficiency of chromophore maturation was calculated based on the concentrations of matured and overall GFP.
The extinction coefficients of each sample at 397 nm (ε397) and 495 nm (ε495) were determined using Beer’s Law: A = ε × l × c, where A is experimentally determined absorbance, l is path length and c is the concentration of matured GFPs (= total GFP concentration × efficiency of chromophore maturation).
For the measurement of quantum yield, all protein samples were diluted to an OD397 of 0.100 and then further diluted 100-fold with water. The emission spectra from 450 nm to 600 nm were scanned using an excitation wavelength of 397 nm. The quantum yield of wtGPFUV has been defined in a previous study as 0.79 . The quantum yield of mutants was calculated by comparing their integrated area of emission spectra with that of wtGPFUV. Three experimental replicates were performed for each measurement. The instrumental error was estimated to be 2 %.
Kinetic refolding experiments
To characterize the refolding ability of the deletion mutants, protein samples were denatured in the following solutions: 20 mM Tris–HCl, pH 7.5, 100 mM NaCl, 1 mM ethylenediaminetetraacetic acid (EDTA), 1 mM DTT, 0.20 mg/mL protein and 6 M guanidine hydrochloride (GdnHCl). Protein unfolding was processed at 25 °C for 24 h. Refolding process was initiated by diluting the denaturation solution 20 fold using the same buffer without GdnHCl. The fluorescence recovery was monitored at 25 °C for 60 min. The refolding data were fitted into a double exponential equation with a parallel refolding model using Prism 4.0 (GraphPad Software Inc., La Jolla, CA).
Fluorescence rescue by folding-enhancing mutations
Mutations F64L  and S30R  were introduced into wtGFPUV and deletion mutants by site-directed mutagenesis (see Additional file 4: Table S2 for primers). The whole-cell fluorescence of F64L- or S30R-rescued mutants was assayed as described above. Mutants with double internal deletions were constructed using site-directed mutagenesis and mutation F64L was then introduced into the double deletion mutants. The rescued whole-cell fluorescence was assayed at 20 °C as described above.