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==Riboregulators== | ==Riboregulators== | ||
New insights into this regulatory role of RNA have allowed for engineering new RNA molecules which control cell behavior interactions with other RNAs or with small molecule ligands. One such engineering approach which enables post-transcriptional control of gene expression is the riboregulator developed by Collins and coworkers. The riboregulator is composed of two interacting parts, a cis-repressive sequence and a trans-activating sequence. The cis sequence is placed directly upstream of the ribosome binding site (RBS) of the gene regulated by the riboregulator and is complimentary to the RBS. This sequence forms a stem-loop structure in the 5’-untranslated region of the mRNA which prevents ribosome binding and thus translation. The trans RNA is complementary to the cis sequence. When the trans RNA is present, it binds to the cis sequence, the RBS site becomes exposed, and the ribosome can bind and translate the mRNA. | New insights into this regulatory role of RNA have allowed for engineering new RNA molecules which control cell behavior interactions with other RNAs or with small molecule ligands. One such engineering approach which enables post-transcriptional control of gene expression is the riboregulator developed by Collins and coworkers. The riboregulator is composed of two interacting parts, a cis-repressive sequence and a trans-activating sequence. The cis sequence is placed directly upstream of the ribosome binding site (RBS) of the gene regulated by the riboregulator and is complimentary to the RBS. This sequence forms a stem-loop structure in the 5’-untranslated region of the mRNA which prevents ribosome binding and thus translation. The trans RNA is complementary to the cis sequence. When the trans RNA is present, it binds to the cis sequence, the RBS site becomes exposed, and the ribosome can bind and translate the mRNA. | ||
[[Image:Cis trans diagram.JPG|left|thumb|500px|Figure adopted from: Isaacs FJ, Dwyer DJ, Ding C, Pervouchine DD, Cantor CR, and Collins JJ. Engineered riboregulators enable post-transcriptional control of gene expression. Nat Biotechnol 2004 Jul; 22(7) 841-7.]] | |||
==Riboregulator Integration in Our Phage/''E. coli'' System== | ==Riboregulator Integration in Our Phage/''E. coli'' System== | ||
The riboregulator determines what happens to the cell once it is infected by phage. We aimed to design three types of riboregulated systems (one for each protein N, Q, and Cro). For instance, for the N protein-regulated system, the phage genome would have a mutant version of the N gene as well as an N gene suppressed with a cis-repressive sequence would be additionally integrated into the genome. When the phage infects a wild-type E.coli cell, nothing should happen. However, when it infects a cell with the trans-activating noncoding RNA, the cell should lyse because the RBS of the N gene is no longer sequestered by the cis-repressed RNA loop. | The riboregulator determines what happens to the cell once it is infected by phage. We aimed to design three types of riboregulated systems (one for each protein N, Q, and Cro). For instance, for the N protein-regulated system, the phage genome would have a mutant version of the N gene as well as an N gene suppressed with a cis-repressive sequence would be additionally integrated into the genome. When the phage infects a wild-type E.coli cell, nothing should happen. However, when it infects a cell with the trans-activating noncoding RNA, the cell should lyse because the RBS of the N gene is no longer sequestered by the cis-repressed RNA loop. | ||
==Riboregulator Design== | ==Riboregulator Design== | ||
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[[Image: | [[Image:Wiki overview1.JPG|left|thumb|500px|The general structure of a cis-repressive element for the YFP gene in our inducible Ptet/TetR system.]] | ||
