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A modular strategy for engineering orthogonal chimeric RNA transcription regulators.

Takahashi MK, Lucks JB - Nucleic Acids Res. (2013)

Bottom Line: A comprehensive orthogonality test of these culminated in a 7 × 7 matrix of mutually orthogonal regulators.A comparison between all chimeras tested led to design principles that will facilitate further engineering of orthogonal RNA transcription regulators, and may help elucidate general principles of non-coding RNA regulation.We anticipate that our strategy will accelerate the development of even larger families of orthogonal RNA transcription regulators, and thus create breakthroughs in our ability to construct increasingly sophisticated RNA genetic circuitry.

View Article: PubMed Central - PubMed

Affiliation: School of Chemical and Biomolecular Engineering, Cornell University, Ithaca, NY 14853, USA.

ABSTRACT
Antisense RNA transcription attenuators are a key component of the synthetic biology toolbox, with their ability to serve as building blocks for both signal integration logic circuits and transcriptional cascades. However, a central challenge to building more sophisticated RNA genetic circuitry is creating larger families of orthogonal attenuators that function independently of each other. Here, we overcome this challenge by developing a modular strategy to create chimeric fusions between the engineered transcriptional attenuator from plasmid pT181 and natural antisense RNA translational regulators. Using in vivo gene expression assays in Escherichia coli, we demonstrate our ability to create chimeric attenuators by fusing sequences from five different translational regulators. Mutagenesis of these functional attenuators allowed us to create a total of 11 new chimeric attenutaors. A comprehensive orthogonality test of these culminated in a 7 × 7 matrix of mutually orthogonal regulators. A comparison between all chimeras tested led to design principles that will facilitate further engineering of orthogonal RNA transcription regulators, and may help elucidate general principles of non-coding RNA regulation. We anticipate that our strategy will accelerate the development of even larger families of orthogonal RNA transcription regulators, and thus create breakthroughs in our ability to construct increasingly sophisticated RNA genetic circuitry.

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Design and testing of loop–linear attenuator fusions. (A) Predicted MFE structure from the TransSysI—IS10 (28) translational loop–linear regulator antisense RNA hairpin. Dashed lines represent the cutoff for RNA sequences used to design fusions onto the pT181 attenuator sense hairpin at positions G26 and A24 (Figure 3A). (B) Chimera design for Fusion 13 along with antisense RNA sequence and predicted binding to the attenuator. (C) Average in vivo fluorescence from cells with (gray) or without (white) cognate antisense RNA for Fusion 13 in comparison with the pT181 attenuator. Dashed lines are drawn at the pT181 fluorescence levels throughout. (D) The Fusion 13 OFF level was optimized by switching terminators from TrrnB to t500 (36) on the antisense plasmid. Cartoon shows plasmid architectures and lengths of the antisense and terminator sequences. Average in vivo fluorescence from cells with antisense-TrrnB (gray), antisense-t500 (black) and without (white) cognate antisense. (E) The ON level was optimized by changing fusion sequence length. Fusions 14 and 15 were engineered by replacing the pT181 sequence above position A24 with sequences from TransSysI denoted by dashed lines at I2 and I3, respectively. Average in vivo fluorescence data for these fusions using the antisense-t500 construct as in (C). (F) Predicted MFE structure for the TransSysH [hok/sok, plasmid R1 (37)] translational loop–linear regulatory hairpin. Average in vivo fluorescence data for Fusion 16 created by using the indicated TransSys H sequence (dashed line) at the A24 position in the pT181 hairpin, and using the corresponding antisense-t500 construct. Averages are plotted with error bars representing the standard deviation from measurements of at least five independent transformants.
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gkt452-F4: Design and testing of loop–linear attenuator fusions. (A) Predicted MFE structure from the TransSysI—IS10 (28) translational loop–linear regulator antisense RNA hairpin. Dashed lines represent the cutoff for RNA sequences used to design fusions onto the pT181 attenuator sense hairpin at positions G26 and A24 (Figure 3A). (B) Chimera design for Fusion 13 along with antisense RNA sequence and predicted binding to the attenuator. (C) Average in vivo fluorescence from cells with (gray) or without (white) cognate antisense RNA for Fusion 13 in comparison with the pT181 attenuator. Dashed lines are drawn at the pT181 fluorescence levels throughout. (D) The Fusion 13 OFF level was optimized by switching terminators from TrrnB to t500 (36) on the antisense plasmid. Cartoon shows plasmid architectures and lengths of the antisense and terminator sequences. Average in vivo fluorescence from cells with antisense-TrrnB (gray), antisense-t500 (black) and without (white) cognate antisense. (E) The ON level was optimized by changing fusion sequence length. Fusions 14 and 15 were engineered by replacing the pT181 sequence above position A24 with sequences from TransSysI denoted by dashed lines at I2 and I3, respectively. Average in vivo fluorescence data for these fusions using the antisense-t500 construct as in (C). (F) Predicted MFE structure for the TransSysH [hok/sok, plasmid R1 (37)] translational loop–linear regulatory hairpin. Average in vivo fluorescence data for Fusion 16 created by using the indicated TransSys H sequence (dashed line) at the A24 position in the pT181 hairpin, and using the corresponding antisense-t500 construct. Averages are plotted with error bars representing the standard deviation from measurements of at least five independent transformants.

Mentions: The copy number control element of the insertion sequence IS10 (28) (TransSysI) was chosen as the initial candidate for creating loop–linear chimeric attenuators. Previous work highlighted the ability to use rational design principles to create orthogonal translational regulators from this system (7), which uses a structured antisense RNA molecule to bind to an unstructured target transcript containing the ribosome-binding site. Because the structured pT181 attenuator hairpin is essential for function, we needed to use the structured antisense hairpin from TransSysI as a source of fusion sequence. Therefore, we switched what the natural system uses as antisense and target, resulting in an unstructured antisense for these chimeras (Figure 1E and 4A).Figure 4.


A modular strategy for engineering orthogonal chimeric RNA transcription regulators.

Takahashi MK, Lucks JB - Nucleic Acids Res. (2013)

Design and testing of loop–linear attenuator fusions. (A) Predicted MFE structure from the TransSysI—IS10 (28) translational loop–linear regulator antisense RNA hairpin. Dashed lines represent the cutoff for RNA sequences used to design fusions onto the pT181 attenuator sense hairpin at positions G26 and A24 (Figure 3A). (B) Chimera design for Fusion 13 along with antisense RNA sequence and predicted binding to the attenuator. (C) Average in vivo fluorescence from cells with (gray) or without (white) cognate antisense RNA for Fusion 13 in comparison with the pT181 attenuator. Dashed lines are drawn at the pT181 fluorescence levels throughout. (D) The Fusion 13 OFF level was optimized by switching terminators from TrrnB to t500 (36) on the antisense plasmid. Cartoon shows plasmid architectures and lengths of the antisense and terminator sequences. Average in vivo fluorescence from cells with antisense-TrrnB (gray), antisense-t500 (black) and without (white) cognate antisense. (E) The ON level was optimized by changing fusion sequence length. Fusions 14 and 15 were engineered by replacing the pT181 sequence above position A24 with sequences from TransSysI denoted by dashed lines at I2 and I3, respectively. Average in vivo fluorescence data for these fusions using the antisense-t500 construct as in (C). (F) Predicted MFE structure for the TransSysH [hok/sok, plasmid R1 (37)] translational loop–linear regulatory hairpin. Average in vivo fluorescence data for Fusion 16 created by using the indicated TransSys H sequence (dashed line) at the A24 position in the pT181 hairpin, and using the corresponding antisense-t500 construct. Averages are plotted with error bars representing the standard deviation from measurements of at least five independent transformants.
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gkt452-F4: Design and testing of loop–linear attenuator fusions. (A) Predicted MFE structure from the TransSysI—IS10 (28) translational loop–linear regulator antisense RNA hairpin. Dashed lines represent the cutoff for RNA sequences used to design fusions onto the pT181 attenuator sense hairpin at positions G26 and A24 (Figure 3A). (B) Chimera design for Fusion 13 along with antisense RNA sequence and predicted binding to the attenuator. (C) Average in vivo fluorescence from cells with (gray) or without (white) cognate antisense RNA for Fusion 13 in comparison with the pT181 attenuator. Dashed lines are drawn at the pT181 fluorescence levels throughout. (D) The Fusion 13 OFF level was optimized by switching terminators from TrrnB to t500 (36) on the antisense plasmid. Cartoon shows plasmid architectures and lengths of the antisense and terminator sequences. Average in vivo fluorescence from cells with antisense-TrrnB (gray), antisense-t500 (black) and without (white) cognate antisense. (E) The ON level was optimized by changing fusion sequence length. Fusions 14 and 15 were engineered by replacing the pT181 sequence above position A24 with sequences from TransSysI denoted by dashed lines at I2 and I3, respectively. Average in vivo fluorescence data for these fusions using the antisense-t500 construct as in (C). (F) Predicted MFE structure for the TransSysH [hok/sok, plasmid R1 (37)] translational loop–linear regulatory hairpin. Average in vivo fluorescence data for Fusion 16 created by using the indicated TransSys H sequence (dashed line) at the A24 position in the pT181 hairpin, and using the corresponding antisense-t500 construct. Averages are plotted with error bars representing the standard deviation from measurements of at least five independent transformants.
Mentions: The copy number control element of the insertion sequence IS10 (28) (TransSysI) was chosen as the initial candidate for creating loop–linear chimeric attenuators. Previous work highlighted the ability to use rational design principles to create orthogonal translational regulators from this system (7), which uses a structured antisense RNA molecule to bind to an unstructured target transcript containing the ribosome-binding site. Because the structured pT181 attenuator hairpin is essential for function, we needed to use the structured antisense hairpin from TransSysI as a source of fusion sequence. Therefore, we switched what the natural system uses as antisense and target, resulting in an unstructured antisense for these chimeras (Figure 1E and 4A).Figure 4.

Bottom Line: A comprehensive orthogonality test of these culminated in a 7 × 7 matrix of mutually orthogonal regulators.A comparison between all chimeras tested led to design principles that will facilitate further engineering of orthogonal RNA transcription regulators, and may help elucidate general principles of non-coding RNA regulation.We anticipate that our strategy will accelerate the development of even larger families of orthogonal RNA transcription regulators, and thus create breakthroughs in our ability to construct increasingly sophisticated RNA genetic circuitry.

View Article: PubMed Central - PubMed

Affiliation: School of Chemical and Biomolecular Engineering, Cornell University, Ithaca, NY 14853, USA.

ABSTRACT
Antisense RNA transcription attenuators are a key component of the synthetic biology toolbox, with their ability to serve as building blocks for both signal integration logic circuits and transcriptional cascades. However, a central challenge to building more sophisticated RNA genetic circuitry is creating larger families of orthogonal attenuators that function independently of each other. Here, we overcome this challenge by developing a modular strategy to create chimeric fusions between the engineered transcriptional attenuator from plasmid pT181 and natural antisense RNA translational regulators. Using in vivo gene expression assays in Escherichia coli, we demonstrate our ability to create chimeric attenuators by fusing sequences from five different translational regulators. Mutagenesis of these functional attenuators allowed us to create a total of 11 new chimeric attenutaors. A comprehensive orthogonality test of these culminated in a 7 × 7 matrix of mutually orthogonal regulators. A comparison between all chimeras tested led to design principles that will facilitate further engineering of orthogonal RNA transcription regulators, and may help elucidate general principles of non-coding RNA regulation. We anticipate that our strategy will accelerate the development of even larger families of orthogonal RNA transcription regulators, and thus create breakthroughs in our ability to construct increasingly sophisticated RNA genetic circuitry.

Show MeSH
Related in: MedlinePlus