Journal: Nucleic Acids Research

Article Title: Synthetic negative feedback circuits using engineered small RNAs

doi: 10.1093/nar/gky828

Figure Lengend Snippet: Testing of two synthetic sRNAs targeting the tetR-sfGFP mRNA, and investigating the inducibility of an sRNA. ( A ) Plasmids encoding sRNAs targeting the Shine Dalgarno sequence (pCK209) or start codon (pCK220) of the tetR-sfGFP mRNA were designed and constructed and the ability of each sRNA to inhibit translation assessed. E. coli strain JW3876 was transformed with pCK210 (encoding the autorepressor) and one of pCK209, pCK220 or pAH23 (negative control, no sRNA), cultured at 37°C in EZ rich defined medium supplemented with glycerol and increasing concentrations of aTc, and GFP fluorescence measured at late exponential phase (5 h) by flow cytometry. ( B ) Comparison of the 0 ng/ml aTc samples from A. ( C ) Experimentally-obtained coefficients of variation around the TetR-sfGFP mean from A. ( D ) To test the inducible expression of sRNA, the proD promoter of pCK209 was replaced with the E. coli rhaBAD promoter, resulting in plasmid pAH17. E. coli strain JW3876 was transformed with pCK210 and pAH17, cultured at 37°C in EZ rich defined medium supplemented with glycerol and 0 or 0.4 mg/ml l -rhamnose, and GFP fluorescence measured at late exponential phase (5 h) by flow cytometry. Fluorescence intensity represents the geometric mean of fluorescence. Error bars shown represent the standard deviation of three independent biological replicates. Statistical significance was determined using a one-way ANOVA, followed by a Tukey's multiple comparison test assuming unequal variance.

Article Snippet: A FLUOstar Omega platereader (BMG Labtech) was used for all absorbance measurements of optical density (OD) at 600 nm and GFP fluorescence measured using an Attune NXT flow cytometer (ThermoFisher Scientific).

Techniques: Sequencing, Construct, Transformation Assay, Negative Control, Cell Culture, Fluorescence, Flow Cytometry, Expressing, Plasmid Preparation, Standard Deviation

Journal: Nucleic Acids Research

Article Title: Synthetic negative feedback circuits using engineered small RNAs

doi: 10.1093/nar/gky828

Figure Lengend Snippet: Testing the tunability of the sRNA-tuned autorepressor plasmid pCK221. A ) Schematic diagram of plasmid pCK221. ( B ) Testing the tunability of sRNA translation inhibition with increasing concentrations of L-rhamnose. E. coli strain JW3876 was transformed with pCK221, cultured at 37°C in EZ rich defined medium supplemented with glycerol and increasing concentrations of l -rhamnose and GFP fluorescence measured at late exponential phase (5 h) by flow cytometry. ( C ) Testing the ability to fine-tune the coarse aTc input–output dial with the inducible expression of the sRNA. E. coli strain JW3876 was transformed with pCK221, cultured at 37°C in EZ rich defined medium supplemented with glycerol and increasing concentrations of l -rhamnose and aTc and GFP fluorescence measured at late exponential phase (5 h) by flow cytometry (white columns). Simulated steady-state output is overlayed to show model fit (blue X). ( D ) Experimentally-obtained coefficients of variation around the TetR-sfGFP mean when output is tuned using both l -rhamnose and aTc (white columns) with predicted coefficients of variation overlayed (red stars). Fluorescence intensity represents the geometric mean of fluorescence. Error bars shown represent the standard deviation of three independent biological replicates. Statistical significance was determined using a one-way ANOVA, followed by a Tukey's multiple comparison test assuming unequal variance.

Article Snippet: A FLUOstar Omega platereader (BMG Labtech) was used for all absorbance measurements of optical density (OD) at 600 nm and GFP fluorescence measured using an Attune NXT flow cytometer (ThermoFisher Scientific).

Techniques: Plasmid Preparation, Inhibition, Transformation Assay, Cell Culture, Fluorescence, Flow Cytometry, Expressing, Standard Deviation

Journal: Nucleic Acids Research

Article Title: Synthetic negative feedback circuits using engineered small RNAs

doi: 10.1093/nar/gky828

Figure Lengend Snippet: Characterisation of a closed-loop negative sRNA feedback circuit. ( A ) Plasmids allowing the inducible expression of sRNAs targeting the Shine Dalgarno sequence (pAH15) or start codon (pAH16) of the rhaS-sfGFP mRNA were designed and constructed and the ability of each sRNA to inhibit translation assessed. E. coli strain JW3876 was transformed with pAH12 (constitutive expression of rhaS-sfGFP ) and one of pAH15, pAH16 or pAH23 (negative control, no sRNA), cultured at 37°C in EZ rich defined medium supplemented with glycerol and 0.2 mg/ml l -rhamnose, and GFP fluorescence measured at late exponential phase (5 h) by flow cytometry. ( B ) The tunability of sRNA translation inhibition with increasing concentrations of l -rhamnose was tested. E. coli strain JW3876 was transformed with pCK222 (constitutive expression of rhaS-sfGFP and the P rhaBAD -SD sRNA construct), cultured at 37°C in EZ rich defined medium supplemented with glycerol and increasing concentrations of l -rhamnose, and GFP fluorescence measured at late exponential phase (5 h) by flow cytometry. ( C ) Schematic diagram of plasmid pCK227 where the proD promoter in front of rhaS-sfGFP on pCK222 is replaced by xylS and the Pm promoter from Pseudomonas putida . ( D ) Testing the ability to set the output level with one external input and the feedback strength with another external input. E. coli strain JW3876 was transformed with pCK227, cultured at 37°C in EZ rich defined medium supplemented with glycerol and increasing concentrations of l -rhamnose and m -toluic acid, and GFP fluorescence measured at late exponential phase (5 h) by flow cytometry (white columns). Simulated steady-state output is overlayed to test model fit (blue X). ( E ) Experimentally-obtained coefficients of variation around the TetR-sfGFP mean when output is tuned using both l -rhamnose and m -toluic acid (white columns) with predicted coefficients of variation overlayed (red stars). Fluorescence intensity represents the geometric mean of fluorescence. Error bars shown represent the standard deviation of three independent biological replicates. Statistical significance was determined using a one-way ANOVA, followed by a Tukey's multiple comparison test assuming unequal variance.

Article Snippet: A FLUOstar Omega platereader (BMG Labtech) was used for all absorbance measurements of optical density (OD) at 600 nm and GFP fluorescence measured using an Attune NXT flow cytometer (ThermoFisher Scientific).

Techniques: Expressing, Sequencing, Construct, Transformation Assay, Negative Control, Cell Culture, Fluorescence, Flow Cytometry, Inhibition, Plasmid Preparation, Standard Deviation