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 . 
METHODS
Preparation of RNA sensors and mutants
Linear dose-response curve measurements
Cloning sensors for expression in 
Live cell imaging of the streptavidin sensor
Live cell imaging of MS2 coat protein synthesis
Quantitation of MCP and MCP sensor concentrations
Supplementary Material