Team:Uppsala University/Translational
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+ | <div id="headertext">Silencing sRNA</div> | ||
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We managed to engineer artificial small RNA (sRNA) inhibiting the translation of the antibiotic resistance gene AAC(6’)Ib-cr isolated from a multiresistant bacterial outbreak in a hospital in Sweden. In the process, we managed to demonstrate a standardized method for construction and screening for sRNA successfully against a target mRNA. In practice, sRNA induced silencing of any gene of interest. <br><br> | We managed to engineer artificial small RNA (sRNA) inhibiting the translation of the antibiotic resistance gene AAC(6’)Ib-cr isolated from a multiresistant bacterial outbreak in a hospital in Sweden. In the process, we managed to demonstrate a standardized method for construction and screening for sRNA successfully against a target mRNA. In practice, sRNA induced silencing of any gene of interest. <br><br> | ||
<a href="http://partsregistry.org/Part:BBa_K864444">BBa_K864444</a> is our template target part in which the gene of interest should be inserted. | <a href="http://partsregistry.org/Part:BBa_K864444">BBa_K864444</a> is our template target part in which the gene of interest should be inserted. | ||
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<b>Silencing Truncated AAC(6') by use of fluorescent reporter</b><br> | <b>Silencing Truncated AAC(6') by use of fluorescent reporter</b><br> | ||
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<a href="https://static.igem.org/mediawiki/2012/d/db/Graph_downregulation_data_low.png"><img src="https://static.igem.org/mediawiki/2012/d/db/Graph_downregulation_data_low.png" width="300"></a> | <a href="https://static.igem.org/mediawiki/2012/d/db/Graph_downregulation_data_low.png"><img src="https://static.igem.org/mediawiki/2012/d/db/Graph_downregulation_data_low.png" width="300"></a> | ||
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IntaRNA, an RNA-RNA-interaction prediction software adapted for sRNA and ncRNA interactions [1] was used to predict the sRNA-mRNA interactions of the candidate sRNAs against the target mRNA containing the fusion between AAC(6’)-5’UTR. See <a href="https://2012.igem.org/Team:Uppsala_University/Modelling">modelling page</a> for details. Some of the sRNAs corresponding to the highest SYFP2 downregulation showed a significant basepair matching close to the RBS of the AAC(6’)-5’UTR. A few of the sRNAs was predicted to hybridize in the SYFP2 region of the mRNA.</p><br><br> | IntaRNA, an RNA-RNA-interaction prediction software adapted for sRNA and ncRNA interactions [1] was used to predict the sRNA-mRNA interactions of the candidate sRNAs against the target mRNA containing the fusion between AAC(6’)-5’UTR. See <a href="https://2012.igem.org/Team:Uppsala_University/Modelling">modelling page</a> for details. Some of the sRNAs corresponding to the highest SYFP2 downregulation showed a significant basepair matching close to the RBS of the AAC(6’)-5’UTR. A few of the sRNAs was predicted to hybridize in the SYFP2 region of the mRNA.</p><br><br> | ||
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The E-test with a minimal F-plasmid containing the whole AAC(6’)Ib-cr gene co-expressed with sRNA. A downregulation from MIC>256µg/ml to MIC=53±9µg/ml was the largest measured. Measurement of control and clone 17,37,55 are all triplicates. | The E-test with a minimal F-plasmid containing the whole AAC(6’)Ib-cr gene co-expressed with sRNA. A downregulation from MIC>256µg/ml to MIC=53±9µg/ml was the largest measured. Measurement of control and clone 17,37,55 are all triplicates. | ||
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<p><b>Future perspectives</b> | <p><b>Future perspectives</b> | ||
Our goal is to show the world that there are means of controlling antibiotic resistance in a different manner. One way is to use our small RNA in gene therapy, for example using a phage or conjugative plasmid system.</p> | Our goal is to show the world that there are means of controlling antibiotic resistance in a different manner. One way is to use our small RNA in gene therapy, for example using a phage or conjugative plasmid system.</p> | ||
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[1] Sharma, V., Yamamura, A., Yokobayashi, Y., 2012. Engineering Artificial Small RNAs for Conditional Gene Silencing in Escherichia coli. ACS Synth. Biol. 1, 6–13. <br> | [1] Sharma, V., Yamamura, A., Yokobayashi, Y., 2012. Engineering Artificial Small RNAs for Conditional Gene Silencing in Escherichia coli. ACS Synth. Biol. 1, 6–13. <br> | ||
[2] Holmqvist, E., Unoson, C., Reimegård, J., Wagner, E.G.H., 2012. A mixed double negative feedback loop between the sRNA MicF and the global regulator Lrp. Molecular Microbiology 84, 414–427. | [2] Holmqvist, E., Unoson, C., Reimegård, J., Wagner, E.G.H., 2012. A mixed double negative feedback loop between the sRNA MicF and the global regulator Lrp. Molecular Microbiology 84, 414–427. | ||
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Revision as of 16:30, 26 October 2012
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