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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 two additional loop–loop chimeric attenuator systems. (A) Predicted MFE structures of the first hairpin from the pT181 transcription attenuator and the analogous hairpins from translational loop–loop regulators from plasmids: R1—TransSysR (34), and ColIB-P9—TransSysC (35). Numbers marking the pT181 structure represent the base number in the attenuator sequence starting at the 5′ end. Dashed lines represent the fusion position on the pT181 hairpin with sequences indicated from TransSysR and TransSysC. RNA sequences from TransSysR (Fusion 4) and TransSysC (Fusion 5) replaced the pT181 sequence above the dashed line at pT181 position G26. TransSysC sequence replaced the pT181 sequence above the dashed line at pT181 position A24 (Fusion 6). (B) Average in vivo fluorescence from cells with (gray) or without (white) cognate antisense RNA. Dashed lines are drawn at the pT181 fluorescence levels. Percent attenuation values (OFF level) are noted on the plot. Averages are plotted with error bars representing the standard deviation from measurements of at least five independent transformants.
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gkt452-F3: Design and testing of two additional loop–loop chimeric attenuator systems. (A) Predicted MFE structures of the first hairpin from the pT181 transcription attenuator and the analogous hairpins from translational loop–loop regulators from plasmids: R1—TransSysR (34), and ColIB-P9—TransSysC (35). Numbers marking the pT181 structure represent the base number in the attenuator sequence starting at the 5′ end. Dashed lines represent the fusion position on the pT181 hairpin with sequences indicated from TransSysR and TransSysC. RNA sequences from TransSysR (Fusion 4) and TransSysC (Fusion 5) replaced the pT181 sequence above the dashed line at pT181 position G26. TransSysC sequence replaced the pT181 sequence above the dashed line at pT181 position A24 (Fusion 6). (B) Average in vivo fluorescence from cells with (gray) or without (white) cognate antisense RNA. Dashed lines are drawn at the pT181 fluorescence levels. Percent attenuation values (OFF level) are noted on the plot. Averages are plotted with error bars representing the standard deviation from measurements of at least five independent transformants.

Mentions: We then sought to expand our family of chimeric attenuators using the findings from the TransSysM fusions. Two additional loop–loop translational regulators were then chosen from plasmids R1 (34) (TransSysR) and ColIB-P9 (35) (TransSysC). Because the only working chimera from TransSysM included a predicted interior loop structure above position G26, we sought to preserve this feature in the designs of the next set of loop–loop chimeras. The TransSysR and TransSysC hairpins are shown in Figure 3A, with fused sequences denoted by dashed lines. Cartoons of the chimeras are shown in Figure 3B along with the expression data, and predicted secondary structures of fusions and corresponding chimeric antisense RNAs are shown in Supplementary Figure S3. Fusion 4 was functional, with an ON level slightly higher than that of pT181 (1.17), and an OFF level of 81%. However, Fusion 5 had a poor OFF level with only 27% attenuation. To address this, we tried a second pT181 fusion position within the previously noted region found to be important for changing antisense/attenuator interaction specificity (10) (above position C21). The same RNA sequence from TransSysC as in Fusion 5 was fused above the base pair at A24 (Figure 3A). This resulted in a functioning attenuator, Fusion 6, with an ON level slightly higher than that of pT181 (1.20) and on OFF level of 82%.Figure 3.


A modular strategy for engineering orthogonal chimeric RNA transcription regulators.

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

Design and testing of two additional loop–loop chimeric attenuator systems. (A) Predicted MFE structures of the first hairpin from the pT181 transcription attenuator and the analogous hairpins from translational loop–loop regulators from plasmids: R1—TransSysR (34), and ColIB-P9—TransSysC (35). Numbers marking the pT181 structure represent the base number in the attenuator sequence starting at the 5′ end. Dashed lines represent the fusion position on the pT181 hairpin with sequences indicated from TransSysR and TransSysC. RNA sequences from TransSysR (Fusion 4) and TransSysC (Fusion 5) replaced the pT181 sequence above the dashed line at pT181 position G26. TransSysC sequence replaced the pT181 sequence above the dashed line at pT181 position A24 (Fusion 6). (B) Average in vivo fluorescence from cells with (gray) or without (white) cognate antisense RNA. Dashed lines are drawn at the pT181 fluorescence levels. Percent attenuation values (OFF level) are noted on the plot. Averages are plotted with error bars representing the standard deviation from measurements of at least five independent transformants.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3753616&req=5

gkt452-F3: Design and testing of two additional loop–loop chimeric attenuator systems. (A) Predicted MFE structures of the first hairpin from the pT181 transcription attenuator and the analogous hairpins from translational loop–loop regulators from plasmids: R1—TransSysR (34), and ColIB-P9—TransSysC (35). Numbers marking the pT181 structure represent the base number in the attenuator sequence starting at the 5′ end. Dashed lines represent the fusion position on the pT181 hairpin with sequences indicated from TransSysR and TransSysC. RNA sequences from TransSysR (Fusion 4) and TransSysC (Fusion 5) replaced the pT181 sequence above the dashed line at pT181 position G26. TransSysC sequence replaced the pT181 sequence above the dashed line at pT181 position A24 (Fusion 6). (B) Average in vivo fluorescence from cells with (gray) or without (white) cognate antisense RNA. Dashed lines are drawn at the pT181 fluorescence levels. Percent attenuation values (OFF level) are noted on the plot. Averages are plotted with error bars representing the standard deviation from measurements of at least five independent transformants.
Mentions: We then sought to expand our family of chimeric attenuators using the findings from the TransSysM fusions. Two additional loop–loop translational regulators were then chosen from plasmids R1 (34) (TransSysR) and ColIB-P9 (35) (TransSysC). Because the only working chimera from TransSysM included a predicted interior loop structure above position G26, we sought to preserve this feature in the designs of the next set of loop–loop chimeras. The TransSysR and TransSysC hairpins are shown in Figure 3A, with fused sequences denoted by dashed lines. Cartoons of the chimeras are shown in Figure 3B along with the expression data, and predicted secondary structures of fusions and corresponding chimeric antisense RNAs are shown in Supplementary Figure S3. Fusion 4 was functional, with an ON level slightly higher than that of pT181 (1.17), and an OFF level of 81%. However, Fusion 5 had a poor OFF level with only 27% attenuation. To address this, we tried a second pT181 fusion position within the previously noted region found to be important for changing antisense/attenuator interaction specificity (10) (above position C21). The same RNA sequence from TransSysC as in Fusion 5 was fused above the base pair at A24 (Figure 3A). This resulted in a functioning attenuator, Fusion 6, with an ON level slightly higher than that of pT181 (1.20) and on OFF level of 82%.Figure 3.

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