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secondary structure prediction was performed using mfold online software . mutated and truncated rnas were created by using single stranded dna templates ( integrated dna technologies ) with the desired mutations or truncations and pcr amplifying these sequences to create double stranded dna templates using primers which included a 5 t7 promoter sequence .
pcr products were then purified with pcr purification columns ( qiagen ) and used as templates for in vitro t7 transcription reactions ( epicentre ) as described previously .
dose - response curves for each sensor in response to the target protein were determined by measuring the increase in fluorescence as a function of increasing target concentration in the presence of a fixed concentration of rna sensor ( 200 - 400 nm ) and a fixed concentration of fluorophore ( 10 m ) .
fluorescence measurements were performed using a perkin elmer ls-55 fluorescence spectrometer using the following instrument parameters : excitation wavelength , 460 nm ; emission wavelength , 501 nm ; slit widths , 10 nm .
the mcp and streptavidin sensors were pcr amplified with primers containing either eagi or sacii restriction sites on the 5 or 3 ends of the sensor sequence , respectively .
the sensors were then cloned into a plasmid containing a chimera of the human trna3 scaffold , which we previously used for spinach and spinach - based metabolite sensors , and which has previously been shown to stabilize heterologous expression of rna aptamers in e. coli .
the entire trna - sensor construct was then pcr amplified with a forward primer containing a bglii site ( italics ) and a t7 promoter sequence ( bold ) : 5- cag tag aga tct taa tac gac tca cta tag gct cga ggc ccg gat agc tca gtc gg -3 , and a reverse primer containing a blpi site ( italics ) : 5- gat cag gct cag ctg gcg ccc gaa cag ggac-3. this pcr product was then cloned into a pet28c vector directly upstream of the t7 terminator sequence with bglii and blpi restriction sites .
bl21 e. coli cells were transformed to harbor either the streptavidin sensor alone in the context of a trna scaffold in pet28c under the control of a t7 promoter as previously described ( pet38c - trna - streptavidin sensor ) or cotransformed to express both pet28c - trna - streptavidin sensor and pet21a - streptavidin - alive .
cells were grown in lb media supplemented with 50 g / ml kanamycin and 100 g / ml ampicillin and 50 g / ml kanamycin , respectively .
sensor and protein synthesis were induced by addition of iptg to 1 mm for 2 h. after induction , cells were adhered and imaged as described below .
dh5 turbo e. coli cells ( new england biolabs ) were used for infection by ms2 phage and imaging because they harbor the f plasmid necessary for ms2 infection .
cells were transformed with 40 ng of plasmid dna expressing the appropriate rna aptamer in the context of a trna scaffold in pet28c under the control of a t7 promoter as previously described .
additionally , since this strain of dh5 does not already contain a genomically incorporated t7 rna polymerase , the streptavidin sensor plasmid was cotransformed with a plasmid harboring the t7 polymerase gene under control of a trc promoter .
cells were grown overnight on lb media supplemented with 100 g / ml ampicillin and 50 g / ml kanamycin to select for those cells that harbored both plasmids .
single colonies were then picked for inoculation in lb supplemented with100 g / ml carbenicilin and 50 g / ml kanamycin and grown at 37c with shaking to od600 = 0.4 , at which point iptg was added to a final concentration of 1 mm to induce t7 polymerase and mcp aptamer expression .
100 l of culture was then removed , spun down to pellet the culture , and resuspended in 2 ml of ph 6.0 m9 minimal media .
a 200 l aliquot of resuspended culture was then plated on pll - coated 24-well glass - bottom dishes ( mattek ) and incubated for 45 minutes at 37c .
adherent cells were washed twice and then incubated with 50 m dfhbi in ph 6.0 m9 minimal media for 1 hour at 25c .
cells were then treated with ms2 phage ( atcc # 15597-b1 ) with an moi of either 10 or 0.1 , as specified .
the titer of infection - competent virus was determined by an agar - overlay plaque - formation assay as previously described .
live fluorescence images were taken with a coolsnap hq2 ccd camera through a 60x oil objective mounted on a nikon te2000 microscope and analyzed with the nis - elements software .
the filter set used was a sputter coated filter cube with excitation filter 470/40 , dichroic mirror 495 ( long pass ) and emission filter 525/50 ( chroma technology ) .
the experiments in which bfp and the mcp sensor were coexpressed were carried out as described above , however , bfp and the mcp sensor were coexpressed from the pet - duet vector ( novagen ) . for experiments where tetracycline was used to inhibit protein synthesis ,
cells were incubated with 10 g / ml tetracycline for 10 minutes before treatment with ms2 phage
dh5 turbo e. coli cells were infected with purified ms2 phage at an moi of 10 . at appropriate time points following infection ,
cells were measured for whole - cell fluorescence using a tecan safireii plate reader or collected and lysed by incubation at 95c for 10 min in sds sample buffer . for fluorescence measurements , background fluorescence for uninfected cells incubated with 50 m dfhbi
was subtracted from all time points . for analysis by western blot , 0.5 od units of e. coli in sample buffer per time point
western blot was carried out using a rabbit polyclonal antibody against mcp ( millipore ) .
an anti - rabbit horse radish peroxidase - conjugated antibody was used to detect signal .
bands from western blot analysis were quantified using image lab software version 4.0 ( bio - rad ) .
the following calculations were used to measure the approximate concentration of mcp molecules synthesized per cell . based on quantification ,
the molecular weight of mcp is 12.5 kda , indicating that 6.2  10 copies of mcp were synthesized per cell after ms2 infection . using the general conversion of 1000 copies per cell equals 1 m
the same conversion was applied to determine the total mcp sensor concentration from northern blot analysis to determine that roughly 120 m sensor is present under these conditions .
northern blot analysis was carried out as previously described , and probled using a 5cy3-labeled probe against the mcp sensor ( 5- ggacccgtccttcaccatttcattcag-3 ) ( idt dna ) .
bands from northern blot analysis were quantified using image lab software version 4.0 ( bio - rad ) .
secondary structure prediction was performed using mfold online software . mutated and truncated rnas were created by using single stranded dna templates ( integrated dna technologies ) with the desired mutations or truncations and pcr amplifying these sequences to create double stranded dna templates using primers which included a 5 t7 promoter sequence .
pcr products were then purified with pcr purification columns ( qiagen ) and used as templates for in vitro t7 transcription reactions ( epicentre ) as described previously .
dose - response curves for each sensor in response to the target protein were determined by measuring the increase in fluorescence as a function of increasing target concentration in the presence of a fixed concentration of rna sensor ( 200 - 400 nm ) and a fixed concentration of fluorophore ( 10 m ) .
fluorescence measurements were performed using a perkin elmer ls-55 fluorescence spectrometer using the following instrument parameters : excitation wavelength , 460 nm ; emission wavelength , 501 nm ; slit widths , 10 nm .
the mcp and streptavidin sensors were pcr amplified with primers containing either eagi or sacii restriction sites on the 5 or 3 ends of the sensor sequence , respectively .
the sensors were then cloned into a plasmid containing a chimera of the human trna3 scaffold , which we previously used for spinach and spinach - based metabolite sensors , and which has previously been shown to stabilize heterologous expression of rna aptamers in e. coli .
the entire trna - sensor construct was then pcr amplified with a forward primer containing a bglii site ( italics ) and a t7 promoter sequence ( bold ) : 5- cag tag aga tct taa tac gac tca cta tag gct cga ggc ccg gat agc tca gtc gg -3 , and a reverse primer containing a blpi site ( italics ) : 5- gat cag gct cag ctg gcg ccc gaa cag ggac-3. this pcr product was then cloned into a pet28c vector directly upstream of the t7 terminator sequence with bglii and blpi restriction sites .
bl21 e. coli cells were transformed to harbor either the streptavidin sensor alone in the context of a trna scaffold in pet28c under the control of a t7 promoter as previously described ( pet38c - trna - streptavidin sensor ) or cotransformed to express both pet28c - trna - streptavidin sensor and pet21a - streptavidin - alive .
cells were grown in lb media supplemented with 50 g / ml kanamycin and 100 g / ml ampicillin and 50 g / ml kanamycin , respectively .
sensor and protein synthesis were induced by addition of iptg to 1 mm for 2 h. after induction , cells were adhered and imaged as described below .
dh5 turbo e. coli cells ( new england biolabs ) were used for infection by ms2 phage and imaging because they harbor the f plasmid necessary for ms2 infection .
cells were transformed with 40 ng of plasmid dna expressing the appropriate rna aptamer in the context of a trna scaffold in pet28c under the control of a t7 promoter as previously described . additionally , since this strain of dh5 does not already contain a genomically incorporated t7 rna polymerase , the streptavidin sensor plasmid was cotransformed with a plasmid harboring the t7 polymerase gene under control of a trc promoter .
cells were grown overnight on lb media supplemented with 100 g / ml ampicillin and 50 g / ml kanamycin to select for those cells that harbored both plasmids .
single colonies were then picked for inoculation in lb supplemented with100 g / ml carbenicilin and 50 g / ml kanamycin and grown at 37c with shaking to od600 = 0.4 , at which point iptg was added to a final concentration of 1 mm to induce t7 polymerase and mcp aptamer expression .
100 l of culture was then removed , spun down to pellet the culture , and resuspended in 2 ml of ph 6.0 m9 minimal media .
a 200 l aliquot of resuspended culture was then plated on pll - coated 24-well glass - bottom dishes ( mattek ) and incubated for 45 minutes at 37c .
adherent cells were washed twice and then incubated with 50 m dfhbi in ph 6.0 m9 minimal media for 1 hour at 25c .
cells were then treated with ms2 phage ( atcc # 15597-b1 ) with an moi of either 10 or 0.1 , as specified .
the titer of infection - competent virus was determined by an agar - overlay plaque - formation assay as previously described .
live fluorescence images were taken with a coolsnap hq2 ccd camera through a 60x oil objective mounted on a nikon te2000 microscope and analyzed with the nis - elements software .
the filter set used was a sputter coated filter cube with excitation filter 470/40 , dichroic mirror 495 ( long pass ) and emission filter 525/50 ( chroma technology ) .
the experiments in which bfp and the mcp sensor were coexpressed were carried out as described above , however , bfp and the mcp sensor were coexpressed from the pet - duet vector ( novagen ) . for experiments where tetracycline was used to inhibit protein synthesis , cells were grown , induced , and adhered to slides as described above .
cells were incubated with 10 g / ml tetracycline for 10 minutes before treatment with ms2 phage
dh5 turbo e. coli cells were infected with purified ms2 phage at an moi of 10 . at appropriate time points following infection ,
cells were measured for whole - cell fluorescence using a tecan safireii plate reader or collected and lysed by incubation at 95c for 10 min in sds sample buffer . for fluorescence measurements , background fluorescence for uninfected cells incubated with 50 m dfhbi
0.5 od units of e. coli in sample buffer per time point was subjected to denaturing polyacrylamide gel electrophoresis .
western blot was carried out using a rabbit polyclonal antibody against mcp ( millipore ) .
an anti - rabbit horse radish peroxidase - conjugated antibody was used to detect signal .
bands from western blot analysis were quantified using image lab software version 4.0 ( bio - rad ) .
the following calculations were used to measure the approximate concentration of mcp molecules synthesized per cell . based on quantification ,
the molecular weight of mcp is 12.5 kda , indicating that 6.2  10 copies of mcp were synthesized per cell after ms2 infection . using the general conversion of 1000 copies per cell equals 1 m
the same conversion was applied to determine the total mcp sensor concentration from northern blot analysis to determine that roughly 120 m sensor is present under these conditions .
northern blot analysis was carried out as previously described , and probled using a 5cy3-labeled probe against the mcp sensor ( 5- ggacccgtccttcaccatttcattcag-3 ) ( idt dna ) .
bands from northern blot analysis were quantified using image lab software version 4.0 ( bio - rad ) .
we show that the difficulties in imaging the dynamics of protein expression in live bacterial cells can be overcome using fluorescent sensors based on spinach , an rna that activates the fluorescence of a small - molecule fluorophore . 
 these rnas selectively bind target proteins , and exhibit fluorescence increases that enable protein expression to be imaged in living cells . 
 these sensors provide a general strategy to image protein expression in single bacteria in real - time .