Team:DTU-Denmark/Project

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[[File:DTU2011_project_fig1.png|200px|thumb|right|Schematic representation of RNA interactions in trap-RNA system. Blue is any target mRNA. Red is sRNA. Green is trap-RNA.]]
[[File:DTU2011_project_fig1.png|200px|thumb|right|Schematic representation of RNA interactions in trap-RNA system. Blue is any target mRNA. Red is sRNA. Green is trap-RNA.]]
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== The experiments ==
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== Experiments ==
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Verifying that the envisioned small RNA based gene silencing is possible. Plasmids containing and strains deleted for the components were constructed. The dynamic range of the araBAD promoter were expanded.
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Verifying that the envisioned small RNA based gene silencing is possible. Plasmids containing and strains deleted for the components were constructed providing a biological model. The dynamic range of the araBAD promoter was expanded.
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The experiments are a proof of concept, showing that the sRNA and the trap-RNA can be used to silence and control gene expression by targeting the chitoporin Shine-Dalgarno. The experiments fall into '''three parts'''. For full description go to [[Team:DTU-Denmark/Project_experiment|experiments]].
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'''Construction of plasmids''' necessary for testing our system involves taking the native system from ''E. coli'', as well as a slightly modified system and putting them on plasmids that let us both control the expression of these components and measure the output of the system.
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'''Strain construction''' involves deleting the original genes from the chromosome of a ''E. coli'' W3110 strain. Since we use the original genes, these need to be deleted from the chromosome to prevent them from interfering with our measurements.
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'''Improving the ''araBAD'' promoter''' entails expanding the dynamic range of this promoter by modifying the -10 and -35 sequence of the promoter, as well as randomly changing the nucleotide sequence around and in between these sequences. Since the ''araBAD'' promoter is used in our project improving this promoter could lead to even finer control of our system.
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== Modeling ==
== Modeling ==

Revision as of 17:41, 20 September 2011

Project

Contents

The abstract

Small regulatory RNA is an active area of research with untapped possibilities for application in biotechnology. A novel type of small RNA regulation displaying favorable properties was investigated ...

Schematic representation of RNA interactions in trap-RNA system. Blue is any target mRNA. Red is sRNA. Green is trap-RNA.

Experiments

Verifying that the envisioned small RNA based gene silencing is possible. Plasmids containing and strains deleted for the components were constructed providing a biological model. The dynamic range of the araBAD promoter was expanded.

Modeling

Kinetic models of the system are the basis for modeling. Blue is target mRNA, red is small RNA and green is trap-RNA

Modeling provides a framework for characterization and the means to incorporate the trap-RNA system into larger models. A steady state analysis revealed that each trap-RNA system has a characteristic fold repression. The influence of parameters on the fold repression was investigated to help guide the design of the trap-RNA system. For more information got to modeling.

Bioinformatic

A bioinformatic study was performed to investigate the possibilities of engineering the trap-RNA system. The aim of engineering being to target any mRNA and to change properties of the system. The study elucidates interesting features of sequence and secondary structure conservation guiding future genetic engineering. For full analysis go to bioinformatic.

References

[1] Datsenko, K.A. & Wanner, B.L. One-step inactivation of chromosomal genes in Escherichia coli K-12 using PCR products. Proceedings of the National Academy of Sciences 97, 6640-6645(2000).

[2] Figueroa-Bossi, Nara, Martina Valentini, Laurette Malleret, and Lionello Bossi. “Caught at its own game: regulatory small RNA inactivated by an inducible transcript mimicking its target.” Genes & Development 23, no. 17 (2009): 2004 -2015.

[3] Hayashi, K. et al. Highly accurate genome sequences of Escherichia coli K-12 strains MG1655 and W3110. Molecular Systems Biology 2, 2006.0007(2006).

[4] Lambert, J.M., Bongers, R.S. & Kleerebezem, M. Cre-lox-based system for multiple gene deletions and selectable-marker removal in Lactobacillus plantarum. Applied and environmental microbiology 73, 1126-35(2007).

[5] Overgaard, Martin, Jesper Johansen, Jakob Møller‐Jensen, and Poul Valentin‐Hansen. “Switching off small RNA regulation with trap‐mRNA.” Molecular Microbiology 73, no. 5 (September 2009): 790-800.


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