Team:Alberta NINT/Project

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**Manipulating RNA attenuator sequences to generate genetic logic circuits in E. coli...**

The integrated circuit was a major milestone of modern technology that facilitated the entrance of society into the information age. Electrical circuits that once occupied an entire room are now able to be placed on a single micro-chip the size of a postage stamp. The implementation of Boolean logic (AND, OR, NOT, etc.) using integrated electronic circuits forms the basis for modern day computers.

When compared to electronics, similar levels of integration have yet to be realized in biological systems. While electronic devices are inherently connectible due to their common currency (flow of electrons), biological circuits lack similar connectivity. This poses a significant challenge to the development of truly programmable biosystems. The Logi-coli[i] project hopes to overcome this challenge by constructing connectible, extensible biological circuits based on antisense RNA regulation of transcriptional attenuation.

Transcriptional attenuation is a common biological mechanism for controlling transcriptional activity. As RNA strands are produced by RNA polymerase transcription of a DNA template, they often form secondary “stem-loop” structures due to base-pair complementarity of inverted repeat sequences. Inverted repeat sequences are short sequences in the transcript that are repeated at a later position in reverse order. These repeat sequences cause the RNA strand to fold back on itself due to base-pairing between complementary A-U and C-G nucleotide pairs in the different repeat regions. A terminator is formed when such a simple stem-loop is followed by a “UUUAUUU” sequence that causes the severing of the nascent RNA strand and the dislocation of the RNA polymerase from the DNA template strand. An attenuator is a terminator structure found at the start of a transcript and has the ability to prematurely truncate the transcript.

Transcriptional attenuation can be modified by designing an anti-sense input RNA strand to disrupt the normal base-pairing in the attenuator stem-loop structure. As a result of this disruption, the RNA polymerase can continue transcription past the attenuator sequence. Using software called RNAstructure, our team hopes to be able to rationally design functional attenuator and corresponding input anti-sense RNA sequences. In this way, we hope to provide a means for connecting series of circuits by using the output RNA strand of one circuit to regulate the transcription of a second circuit. We hope to combine simple attenuators into more complex structures that act as biological equivalents to electronic Boolean logic gates (AND, OR, XOR, etc.)

In regards to the final output, a coding region of LacZ or GFP will be expressed to provide evidence of the functionality of the logic circuits. We hope to connect these basic logic gates into even more complex circuits such as half-adders or full-adders. This important step in the design of complex logic in biological circuits has eluded researchers for a number of years, but we believe our approach to have great potential for success.

Project Details

Coming Soon!

Results

Coming Soon!

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 == '''Manipulating RNA attenuator sequences to generate genetic logic circuits in ''E. coli''...''' ==
-While electronic devices are inherently connectible due to their common currency (flow of electrons), biological circuits are more complex because they lack connectivity.  The Logi-coli[i] project hopes to construct connectible biological circuits based on antisense RNA regulation of transcriptional attenuation.  Transcriptional attenuation is a common prokaryotic mechanism for controlling transcriptional productivity.  As RNA transcripts are produced by the enzyme RNA polymerase moving along the DNA template strand, they often form secondary stem-loop structures.  The terminator is a simple stem-loop followed by the “UUUAUUU” terminator sequence that causes the severing of the nascent RNA strand and the dislocation of RNA polymerase from the DNA template strand.  An attenuator is a terminator structure found at the start of a transcript that causes the transcript to be prematurely truncated. Transcriptional attenuation can be modified by designing an anti-sense input RNA strand to utilize base-pairing with complementary parts of the stem-loop structure of the attenuator to disrupt it.  In this way, RNA polymerase would pass over the attenuator sequence and continue with transcription.  Using software called RNAstructure, our team will be able to rationally design the appropriate input RNA and attenuator sequences to accomplish this.  These specific sequences will interact with specified free energies and base-pair complementarity.  In this way, the output RNA strand of one circuit can be used as the input RNA strand to the next circuit.  This provides a means for connecting a series of circuits.  For the final output, a coding region of LacZ or GFP would be transcribed to provide evidence of the connectivity of the circuits.  Complex circuits such as AND, OR, NOT, EXOR, and NAND gates can be connected together to create half-adders or full-adders.
+The integrated circuit was a major milestone of modern technology that facilitated the entrance of society into the information age. Electrical circuits that once occupied an entire room are now able to be placed on a single micro-chip the size of a postage stamp.  The implementation of Boolean logic (AND, OR, NOT, etc.) using integrated electronic circuits forms the basis for modern day computers.
+When compared to electronics, similar levels of integration have yet to be realized in biological systems.  While electronic devices are inherently connectible due to their common currency (flow of electrons), biological circuits lack similar connectivity.  This poses a significant challenge to the development of truly programmable biosystems.  The Logi-coli[i] project hopes to overcome this challenge by constructing connectible, extensible biological circuits based on antisense RNA regulation of transcriptional attenuation.
+Transcriptional attenuation is a common biological mechanism for controlling transcriptional activity.  As RNA strands are produced by RNA polymerase transcription of a DNA template, they often form secondary “stem-loop” structures due to base-pair complementarity of inverted repeat sequences.  Inverted repeat sequences are short sequences in the transcript that are repeated at a later position in reverse order.  These repeat sequences cause the RNA strand to fold back on itself due to base-pairing between complementary A-U and C-G nucleotide pairs in the different repeat regions.  A terminator is formed when such a simple stem-loop is followed by a “UUUAUUU” sequence that causes the severing of the nascent RNA strand and the dislocation of the RNA polymerase from the DNA template strand.  An attenuator is a terminator structure found at the start of a transcript and has the ability to prematurely truncate the transcript.
+Transcriptional attenuation can be modified by designing an anti-sense input RNA strand to disrupt the normal base-pairing in the attenuator stem-loop structure.  As a result of this disruption, the RNA polymerase can continue transcription past the attenuator sequence.  Using software called RNAstructure, our team hopes to be able to rationally design functional attenuator and corresponding input anti-sense RNA sequences.  In this way, we hope to provide a means for connecting series of circuits by using the output RNA strand of one circuit to regulate the transcription of a second circuit.  We hope to combine simple attenuators into more complex structures that act as biological equivalents to electronic Boolean logic gates (AND, OR, XOR, etc.)
+In regards to the final output, a coding region of LacZ or GFP will be expressed to provide evidence of the functionality of the logic circuits.  We hope to connect these basic logic gates into even more complex circuits such as half-adders or full-adders.  This important step in the design of complex logic in biological circuits has eluded researchers for a number of years, but we believe our approach to have great potential for success.
 == Project Details==