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Quantification of the gene silencing performances of rationally-designed synthetic small RNAs

Abstract

Small RNAs (sRNAs) are genetic tools for the efficient and specific tuning of target genes expression in bacteria. Inspired by naturally occurring sRNAs, recent works proposed the use of artificial sRNAs in synthetic biology for predictable repression of the desired genes. Their potential was demonstrated in several application fields, such as metabolic engineering and bacterial physiology studies. Guidelines for the rational design of novel sRNAs have been recently proposed. According to these guidelines, in this work synthetic sRNAs were designed, constructed and quantitatively characterized in Escherichia coli. An sRNA targeting the reporter gene RFP was tested by measuring the specific gene silencing when RFP was expressed at different transcription levels, under the control of different promoters, in different strains, and in single-gene or operon architecture. The sRNA level was tuned by using plasmids maintained at different copy numbers. Results demonstrated that RFP silencing worked as expected in an sRNA and mRNA expression-dependent fashion. A mathematical model was used to support sRNA characterization and to estimate an efficiency-related parameter that can be used to compare the performance of the designed sRNA. Gene silencing was also successful when RFP was placed in a two-gene synthetic operon, while the non-target gene (GFP) in the operon was not considerably affected. Finally, silencing was evaluated for another designed sRNA targeting the endogenous lactate dehydrogenase gene. The quantitative study performed in this work elucidated interesting performance-related and context-dependent features of synthetic sRNAs that will strongly support predictable gene silencing in disparate basic or applied research studies.

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Correspondence to Paolo Magni.

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Supplementary material 1

Raw data and processed time series for cultures in representative experiments. A Raw absorbance time series of sterile medium (M9) and a non-fluorescent culture (TOP10). B Background-subtracted absorbance (OD 600) time series of a non- luorescent culture (TOP10). C Raw red fluorescence of sterile medium (M9) and a nonfluorescent culture (TOP10) time series, showing that the autofluorescence of bacteria is comparable with the fluorescence of medium and it is not OD 600-dependent. D Raw green fluorescence of sterile medium (M9) and a nonfluorescent culture (TOP10) time series, showing that the autofluorescence of bacteria is higher than the fluorescence of medium and it is OD 600-dependent. E Raw green fluorescence as a function of OD 600 for several non-fluorescent strains assayed in the same experiment; such data are used to compute the OD 600-dependent autofluorescence function (by linear regression), which represents the background green fluorescence at a given OD 600; circles represent data points and solid line represents the regression line. F Raw absorbance time series of three RFP-expressing cultures: Plux-R, sRFP-1A2+Plux-R and J101-R in TOP10. G Background-subtracted OD 600 time series of the three RFPexpressing cultures. H Raw red fluorescence time series of the three RFP-expressing cultures. I Background subtracted red fluorescence time series of the three RFP-expressing cultures, yielding a time series proportional to the total RFP proteins in the microplate well. J Numeric time derivative of RFP divided by OD 600, yielding a signal proportional to the RFP synthesis rate per cell at the steady-state; the time series in the exponential growth phase (OD 600 between 0.05 and 0.18, assumed) is shown for the three RFP-expressing cultures; for each culture, this time series is averaged and divided by the average RFP synthesis rate per cell of the reference culture (see Methods section in the main text). K–O the same time series as panels F–J are shown for two GFP-expressing cultures: Plux-G and sRFP-1A2+Plux-G (PNG 1485 kb)

Supplementary material 2

Doubling times of recombinant strains bearing a single-gene RFP or GFP expression system driven by Plux (Plux-R or Plux-G). A Specific silencing of the target gene (RFP) via sRFP in TOP10 and W. B Unspecific silencing of RFP or GFP via different sRNAs in TOP10 and W. Bars represent the mean doubling time value computed on at least three biological replicates. Error bars represent the 95 % confidence intervals of the mean value (PNG 1350 kb)

Supplementary material 3

Doubling times of recombinant strains bearing an RFP expression system driven by BBa_J23101 (J101-R). Bars represent the mean doubling time value computed on at least three biological replicates in the indicated conditions. Error bars represent the 95 % confidence intervals of the mean value (PNG 285 kb)

Supplementary material 4

Silencing results for RFP expressed by a single-gene cassette (J101-R32) driven by BBa_J23101 with the BBa_B0032 RBS upstream of the RFP gene. A Specific silencing of the target gene (RFP) via sRFP in TOP10. Bars represent the mean Scell value computed on at least three biological replicates. Error bars represent the 95 % confidence intervals of the mean value. Asterisks indicate that the Scell value in the condition is statistically different from the Scell of the expression cassette without sRNAs (J101-R32). Percentages represent the Eff% values. B Doubling times (PNG 509 kb)

Supplementary material 5

Doubling times of recombinant TOP10 bearing the Plux-RG and Plux-GR synthetic operons. A Silencing of the target gene (RFP) and the non-target gene (GFP) via the silencing device sRFP. B Unspecific silencing of RFP and GFP, in the Plux-GR construct, via different sRNAs. Bars represent the mean doubling time value computed on at least three biological replicates. Error bars represent the 95 % confidence intervals of the mean value (PNG 604 kb)

Supplementary material 6

Silencing results for RFP and GFP expressed by the Plux-GR synthetic operon in the W strain. A Silencing of the target gene (RFP) and the non-target gene (GFP) via the silencing device sRFP. B Unspecific silencing of RFP and GFP via different sRNAs. Bars represent the mean Scell value computed on at least three biological replicates. Error bars represent the 95 % confidence intervals of the mean value. Asterisks indicate that the Scell value of RFP or GFP in the condition is statistically different from the Scell of the operon without sRNAs (Plux-GR). Percentages represent the Eff% values. When Scell in a given condition is higher than Scell without sRNA, Eff% value is set to zero (PNG 695 kb)

Supplementary material 7

Doubling times of recombinant W bearing the Plux-GR synthetic operon. A Specific silencing of the target gene (RFP) and the non-target gene (GFP) via the silencing device sRFP. B Unspecific silencing of RFP and GFP via different sRNAs. Bars represent the mean doubling time value computed on at least three biological replicates. Error bars represent the 95 % confidence intervals of the mean value (PNG 576 kb)

Supplementary material 8

Doubling times of recombinant strains bearing the Plux-RG30 and Plux-G30R synthetic operons in TOP10 and W. A Silencing of the target gene (RFP) and the non-target gene (GFP) via the silencing device sRFP. B Unspecific silencing of RFP and GFP via different sRNAs. Bars represent the mean doubling time value computed on at least three biological replicates. Error bars represent the 95 % confidence intervals of the mean value (PNG 1387 kb)

Supplementary material 9

Silencing results for RFP and GFP expressed by the Plux-RG30 and Plux-G30R synthetic operons in TOP10 and W. A Silencing of the target gene (RFP) and the non-target gene (GFP) via the silencing device sRFP. B Unspecific silencing of RFP and GFP via different sRNAs. Bars represent the mean Scell value computed on at least three biological replicates. Error bars represent the 95 % confidence intervals of the mean value. Asterisks indicate that the Scell value of RFP or GFP in the condition is statistically different from the Scell of the operon without sRNA (Plux-RG30 or Plux-G30R). Percentages represent the Eff% values. When Scell in a given condition is higher than Scell without sRNA, Eff% value is set to zero (PNG 2686 kb)

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Massaiu, I., Pasotti, L., Casanova, M. et al. Quantification of the gene silencing performances of rationally-designed synthetic small RNAs. Syst Synth Biol 9, 107–123 (2015). https://doi.org/10.1007/s11693-015-9177-7

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Keywords

  • Small RNA
  • Synthetic biology
  • Quantitative characterization
  • Mathematical modelling
  • Operon
  • Lactate dehydrogenase