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Rapidly characterizing the fast dynamics of RNA genetic circuitry with cell-free transcription-translation (TX-TL) systems.

Takahashi MK, Chappell J, Hayes CA, Sun ZZ, Kim J, Singhal V, Spring KJ, Al-Khabouri S, Fall CP, Noireaux V, Murray RM, Lucks JB - ACS Synth Biol (2014)

Bottom Line: We used this system to measure the response time of an RNA transcription cascade to be approximately five minutes per step of the cascade.We also show that this response time can be adjusted with temperature and regulator threshold tuning.Finally, we use TX-TL to prototype a new RNA network, an RNA single input module, and show that this network temporally stages the expression of two genes in vivo.

View Article: PubMed Central - PubMed

Affiliation: †School of Chemical and Biomolecular Engineering, Cornell University, Ithaca, New York 14850, United States.

ABSTRACT
RNA regulators are emerging as powerful tools to engineer synthetic genetic networks or rewire existing ones. A potential strength of RNA networks is that they may be able to propagate signals on time scales that are set by the fast degradation rates of RNAs. However, a current bottleneck to verifying this potential is the slow design-build-test cycle of evaluating these networks in vivo. Here, we adapt an Escherichia coli-based cell-free transcription-translation (TX-TL) system for rapidly prototyping RNA networks. We used this system to measure the response time of an RNA transcription cascade to be approximately five minutes per step of the cascade. We also show that this response time can be adjusted with temperature and regulator threshold tuning. Finally, we use TX-TL to prototype a new RNA network, an RNA single input module, and show that this network temporally stages the expression of two genes in vivo.

No MeSH data available.


Related in: MedlinePlus

CharacterizingRNA transcriptional attenuators and circuits in TX-TL. (A) Fluorescencetime courses of TX-TL reactions containing the pT181 attenuator reporterplasmid at 0.5 nM, with 8 nM antisense plasmid (+) or 8 nM no-antisensecontrol plasmid (−). (B) SFGFP production rates were calculatedfrom the data in (A) by calculating the slope between consecutivetime points. Boxes represent regions of constant SFGFP production.Blue and red shaded regions in parts A and B represent standard deviationsof at least seven independent reactions performed over multiple dayscalculated at each time point. (C) Average SFGFP production rateswere calculated from the data in boxed regions in part B. Error barsrepresent standard deviations of those averages. The (+) antisensecondition shows 72% attenuation compared to the (−) antisensecondition in TX-TL. (D) Orthogonality of the pT181 attenuator (Att-1)to a pT181 mutant attenuator (Att-2). Average SFGFP production rateswere calculated as in part C. Plots of SFGFP production rates canbe found in Supporting Information Figure S2. Bars represent each attenuator at 0.5 nM with 8 nM of no-antisensecontrol plasmid (blue), pT181 antisense plasmid (AS-1, red), or pT181mutant antisense plasmid (AS-2, purple). (E) Schematic of an RNA transcriptionalcascade. L1 is the same pT181 attenuator (Att-1) reporter plasmidused in parts A–D. In the plasmid for L2, the pT181-mut attenuator(Att-2) regulates two copies of the pT181 antisense (AS-1), each separatedby a ribozyme (triangle).19 The L3 plasmidtranscribes the pT181-mut antisense (AS-2). (F) Average SFGFP productionrates for the three combinations of the transcription cascade depictedin part E. L1 alone (blue bar) leads to high SFGFP production. L1+L2(red bar) results in AS-1 repressing Att-1, thus lower SFGFP production.L1+L2+L3 (purple bar) results in a double inversion leading to highSFGFP production. Total DNA concentration in each reaction was heldconstant at 18.5 nM. In parts D and F, error bars represent standarddeviations from at least seven independent reactions performed overmultiple days.
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fig2: CharacterizingRNA transcriptional attenuators and circuits in TX-TL. (A) Fluorescencetime courses of TX-TL reactions containing the pT181 attenuator reporterplasmid at 0.5 nM, with 8 nM antisense plasmid (+) or 8 nM no-antisensecontrol plasmid (−). (B) SFGFP production rates were calculatedfrom the data in (A) by calculating the slope between consecutivetime points. Boxes represent regions of constant SFGFP production.Blue and red shaded regions in parts A and B represent standard deviationsof at least seven independent reactions performed over multiple dayscalculated at each time point. (C) Average SFGFP production rateswere calculated from the data in boxed regions in part B. Error barsrepresent standard deviations of those averages. The (+) antisensecondition shows 72% attenuation compared to the (−) antisensecondition in TX-TL. (D) Orthogonality of the pT181 attenuator (Att-1)to a pT181 mutant attenuator (Att-2). Average SFGFP production rateswere calculated as in part C. Plots of SFGFP production rates canbe found in Supporting Information Figure S2. Bars represent each attenuator at 0.5 nM with 8 nM of no-antisensecontrol plasmid (blue), pT181 antisense plasmid (AS-1, red), or pT181mutant antisense plasmid (AS-2, purple). (E) Schematic of an RNA transcriptionalcascade. L1 is the same pT181 attenuator (Att-1) reporter plasmidused in parts A–D. In the plasmid for L2, the pT181-mut attenuator(Att-2) regulates two copies of the pT181 antisense (AS-1), each separatedby a ribozyme (triangle).19 The L3 plasmidtranscribes the pT181-mut antisense (AS-2). (F) Average SFGFP productionrates for the three combinations of the transcription cascade depictedin part E. L1 alone (blue bar) leads to high SFGFP production. L1+L2(red bar) results in AS-1 repressing Att-1, thus lower SFGFP production.L1+L2+L3 (purple bar) results in a double inversion leading to highSFGFP production. Total DNA concentration in each reaction was heldconstant at 18.5 nM. In parts D and F, error bars represent standarddeviations from at least seven independent reactions performed overmultiple days.

Mentions: In order to assess the feasibilityof using TX-TL for characterizing RNA circuitry, we first tested thebasic functionality of the central regulator in our RNA cascade, thepT181 transcriptional attenuator19 (Figure 2, Att-1). The pT181 attenuator lies in the 5′-untranslatedregion of a transcript, and functions like a transcriptional switchby either allowing (ON) or blocking (OFF) elongation of RNA polymerase.39,40 The OFF state is induced through an interaction with an antisenseRNA (AS-1), expressed separately in our synthetic context19 (Supporting InformationFigure S1). By transcriptionally fusing the pT181 attenuatorto the super folder green fluorescent protein (SFGFP) coding sequence,we are able to assess functionality of the attenuator by measuringSFGFP fluorescence with and without antisense RNA present (Supporting Information Figure S1).


Rapidly characterizing the fast dynamics of RNA genetic circuitry with cell-free transcription-translation (TX-TL) systems.

Takahashi MK, Chappell J, Hayes CA, Sun ZZ, Kim J, Singhal V, Spring KJ, Al-Khabouri S, Fall CP, Noireaux V, Murray RM, Lucks JB - ACS Synth Biol (2014)

CharacterizingRNA transcriptional attenuators and circuits in TX-TL. (A) Fluorescencetime courses of TX-TL reactions containing the pT181 attenuator reporterplasmid at 0.5 nM, with 8 nM antisense plasmid (+) or 8 nM no-antisensecontrol plasmid (−). (B) SFGFP production rates were calculatedfrom the data in (A) by calculating the slope between consecutivetime points. Boxes represent regions of constant SFGFP production.Blue and red shaded regions in parts A and B represent standard deviationsof at least seven independent reactions performed over multiple dayscalculated at each time point. (C) Average SFGFP production rateswere calculated from the data in boxed regions in part B. Error barsrepresent standard deviations of those averages. The (+) antisensecondition shows 72% attenuation compared to the (−) antisensecondition in TX-TL. (D) Orthogonality of the pT181 attenuator (Att-1)to a pT181 mutant attenuator (Att-2). Average SFGFP production rateswere calculated as in part C. Plots of SFGFP production rates canbe found in Supporting Information Figure S2. Bars represent each attenuator at 0.5 nM with 8 nM of no-antisensecontrol plasmid (blue), pT181 antisense plasmid (AS-1, red), or pT181mutant antisense plasmid (AS-2, purple). (E) Schematic of an RNA transcriptionalcascade. L1 is the same pT181 attenuator (Att-1) reporter plasmidused in parts A–D. In the plasmid for L2, the pT181-mut attenuator(Att-2) regulates two copies of the pT181 antisense (AS-1), each separatedby a ribozyme (triangle).19 The L3 plasmidtranscribes the pT181-mut antisense (AS-2). (F) Average SFGFP productionrates for the three combinations of the transcription cascade depictedin part E. L1 alone (blue bar) leads to high SFGFP production. L1+L2(red bar) results in AS-1 repressing Att-1, thus lower SFGFP production.L1+L2+L3 (purple bar) results in a double inversion leading to highSFGFP production. Total DNA concentration in each reaction was heldconstant at 18.5 nM. In parts D and F, error bars represent standarddeviations from at least seven independent reactions performed overmultiple days.
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fig2: CharacterizingRNA transcriptional attenuators and circuits in TX-TL. (A) Fluorescencetime courses of TX-TL reactions containing the pT181 attenuator reporterplasmid at 0.5 nM, with 8 nM antisense plasmid (+) or 8 nM no-antisensecontrol plasmid (−). (B) SFGFP production rates were calculatedfrom the data in (A) by calculating the slope between consecutivetime points. Boxes represent regions of constant SFGFP production.Blue and red shaded regions in parts A and B represent standard deviationsof at least seven independent reactions performed over multiple dayscalculated at each time point. (C) Average SFGFP production rateswere calculated from the data in boxed regions in part B. Error barsrepresent standard deviations of those averages. The (+) antisensecondition shows 72% attenuation compared to the (−) antisensecondition in TX-TL. (D) Orthogonality of the pT181 attenuator (Att-1)to a pT181 mutant attenuator (Att-2). Average SFGFP production rateswere calculated as in part C. Plots of SFGFP production rates canbe found in Supporting Information Figure S2. Bars represent each attenuator at 0.5 nM with 8 nM of no-antisensecontrol plasmid (blue), pT181 antisense plasmid (AS-1, red), or pT181mutant antisense plasmid (AS-2, purple). (E) Schematic of an RNA transcriptionalcascade. L1 is the same pT181 attenuator (Att-1) reporter plasmidused in parts A–D. In the plasmid for L2, the pT181-mut attenuator(Att-2) regulates two copies of the pT181 antisense (AS-1), each separatedby a ribozyme (triangle).19 The L3 plasmidtranscribes the pT181-mut antisense (AS-2). (F) Average SFGFP productionrates for the three combinations of the transcription cascade depictedin part E. L1 alone (blue bar) leads to high SFGFP production. L1+L2(red bar) results in AS-1 repressing Att-1, thus lower SFGFP production.L1+L2+L3 (purple bar) results in a double inversion leading to highSFGFP production. Total DNA concentration in each reaction was heldconstant at 18.5 nM. In parts D and F, error bars represent standarddeviations from at least seven independent reactions performed overmultiple days.
Mentions: In order to assess the feasibilityof using TX-TL for characterizing RNA circuitry, we first tested thebasic functionality of the central regulator in our RNA cascade, thepT181 transcriptional attenuator19 (Figure 2, Att-1). The pT181 attenuator lies in the 5′-untranslatedregion of a transcript, and functions like a transcriptional switchby either allowing (ON) or blocking (OFF) elongation of RNA polymerase.39,40 The OFF state is induced through an interaction with an antisenseRNA (AS-1), expressed separately in our synthetic context19 (Supporting InformationFigure S1). By transcriptionally fusing the pT181 attenuatorto the super folder green fluorescent protein (SFGFP) coding sequence,we are able to assess functionality of the attenuator by measuringSFGFP fluorescence with and without antisense RNA present (Supporting Information Figure S1).

Bottom Line: We used this system to measure the response time of an RNA transcription cascade to be approximately five minutes per step of the cascade.We also show that this response time can be adjusted with temperature and regulator threshold tuning.Finally, we use TX-TL to prototype a new RNA network, an RNA single input module, and show that this network temporally stages the expression of two genes in vivo.

View Article: PubMed Central - PubMed

Affiliation: †School of Chemical and Biomolecular Engineering, Cornell University, Ithaca, New York 14850, United States.

ABSTRACT
RNA regulators are emerging as powerful tools to engineer synthetic genetic networks or rewire existing ones. A potential strength of RNA networks is that they may be able to propagate signals on time scales that are set by the fast degradation rates of RNAs. However, a current bottleneck to verifying this potential is the slow design-build-test cycle of evaluating these networks in vivo. Here, we adapt an Escherichia coli-based cell-free transcription-translation (TX-TL) system for rapidly prototyping RNA networks. We used this system to measure the response time of an RNA transcription cascade to be approximately five minutes per step of the cascade. We also show that this response time can be adjusted with temperature and regulator threshold tuning. Finally, we use TX-TL to prototype a new RNA network, an RNA single input module, and show that this network temporally stages the expression of two genes in vivo.

No MeSH data available.


Related in: MedlinePlus