Limits...
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.

Show MeSH

Related in: MedlinePlus

Sequence alignment of engineered attenuators. A ClustalW (27) alignment of the 14 attenuators tested for orthogonality. Sequence agreements to the consensus are highlighted by base. The sequence input for the alignment was the first hairpin of each attenuator, including the C-A interior loop of the pT181 sequence at C21 (Figure 2A). Sequences were then separated into two groups: Orthogonal—the eight attenuators from Figure 6B and Non-orthogonal—the remaining six attenuators from Figure 6A. The bold line underlines the six bases that form the predicted loop of the attenuators.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC3753616&req=5

gkt452-F7: Sequence alignment of engineered attenuators. A ClustalW (27) alignment of the 14 attenuators tested for orthogonality. Sequence agreements to the consensus are highlighted by base. The sequence input for the alignment was the first hairpin of each attenuator, including the C-A interior loop of the pT181 sequence at C21 (Figure 2A). Sequences were then separated into two groups: Orthogonal—the eight attenuators from Figure 6B and Non-orthogonal—the remaining six attenuators from Figure 6A. The bold line underlines the six bases that form the predicted loop of the attenuators.

Mentions: One interesting result was the observed orthogonality between the loop–loop chimeras, as they all had the same 6-base loop sequence (5′-TTGGCG-3′). To further examine the sequence determinants of orthogonality amongst our attenuators, we created a sequence alignment of the chimeric region for the 14 attenuators included in our orthogonality matrix test (Figure 7). The sequence input for the alignment included the first hairpin of each attenuator up until the C-A interior loop of the pT181 sequence (at C21) that is present in all attenuators. It is clear from the alignment that the initial interaction at the chimeric attenuator loop alone does not dictate orthogonality. In particular, the loop sequences for five of the orthogonal attenuators are identical, and even similar to loop sequences for attenuators that were found to be in the non-orthogonal group. This is similar to what has been observed in the pT181 system, where combined mutations in the loop and stem region were required to obtain orthogonality (10). The loop–loop fusion sequence alignment does indicate that the main sequence differences between fusions occur in the few bases immediately below the loop and those in the interior loop, which may be the key to orthogonality. Certainly this sequence/function information represents a rich dataset for computational algorithm development to predict functional RNA–RNA interactions, allowing for extensions of this approach to create more orthogonal attenuators.Figure 7.


A modular strategy for engineering orthogonal chimeric RNA transcription regulators.

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

Sequence alignment of engineered attenuators. A ClustalW (27) alignment of the 14 attenuators tested for orthogonality. Sequence agreements to the consensus are highlighted by base. The sequence input for the alignment was the first hairpin of each attenuator, including the C-A interior loop of the pT181 sequence at C21 (Figure 2A). Sequences were then separated into two groups: Orthogonal—the eight attenuators from Figure 6B and Non-orthogonal—the remaining six attenuators from Figure 6A. The bold line underlines the six bases that form the predicted loop of the attenuators.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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

gkt452-F7: Sequence alignment of engineered attenuators. A ClustalW (27) alignment of the 14 attenuators tested for orthogonality. Sequence agreements to the consensus are highlighted by base. The sequence input for the alignment was the first hairpin of each attenuator, including the C-A interior loop of the pT181 sequence at C21 (Figure 2A). Sequences were then separated into two groups: Orthogonal—the eight attenuators from Figure 6B and Non-orthogonal—the remaining six attenuators from Figure 6A. The bold line underlines the six bases that form the predicted loop of the attenuators.
Mentions: One interesting result was the observed orthogonality between the loop–loop chimeras, as they all had the same 6-base loop sequence (5′-TTGGCG-3′). To further examine the sequence determinants of orthogonality amongst our attenuators, we created a sequence alignment of the chimeric region for the 14 attenuators included in our orthogonality matrix test (Figure 7). The sequence input for the alignment included the first hairpin of each attenuator up until the C-A interior loop of the pT181 sequence (at C21) that is present in all attenuators. It is clear from the alignment that the initial interaction at the chimeric attenuator loop alone does not dictate orthogonality. In particular, the loop sequences for five of the orthogonal attenuators are identical, and even similar to loop sequences for attenuators that were found to be in the non-orthogonal group. This is similar to what has been observed in the pT181 system, where combined mutations in the loop and stem region were required to obtain orthogonality (10). The loop–loop fusion sequence alignment does indicate that the main sequence differences between fusions occur in the few bases immediately below the loop and those in the interior loop, which may be the key to orthogonality. Certainly this sequence/function information represents a rich dataset for computational algorithm development to predict functional RNA–RNA interactions, allowing for extensions of this approach to create more orthogonal attenuators.Figure 7.

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