Team:DTU-Denmark/Project

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{{:Team:DTU-Denmark/Templates/Standard_page_begin|Overview}}
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== The abstract ==
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Small regulatory RNA is an active area of research with untapped possibilities for application in biotechnology. We have investigated a novel type of small RNA regulation displaying favorable properties. For longer abstract go to.
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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.]]
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<iframe style="float: right; margin: 15px 0 20px 10px; border: 4px solid white; outline: 1px solid #CCCCCC;" width="440" height="350" src="http://www.youtube.com/embed/dtleYPzOg-Y" frameborder="0" allowfullscreen></iframe>
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== The experiments ==
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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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== Abstract ==
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Small regulatory RNA is an active area of research with untapped possibilities for application in biotechnology. Such applications include convenient gene silencing and fine-tuning of gene expression, which are currently cumbersome processes restricted to well studied bacteria. We have investigated a novel type of RNA regulation based on the [[Team:DTU-Denmark/Background_the_natural_system|chitobiose system]], where the inhibition caused by a small RNA is relieved by another small RNA called trap-RNA.[[File:DTU2011_project_fig1.png|100px|frameless|right|Two-level sRNA regulation. Blue is any target mRNA, red is sRNA and green is trap-RNA.]] We explore the possibility of using the system to uniquely target and repress any gene of interest, potentially providing unprecedented specificity and control of gene silencing. We furthermore constructed araBAD promoters with varying promoter activities using synthetic promoter libraries.
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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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== Experiment: Testing sRNA ==
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[[File:DTU1_Redswap.png|200px|frameless|right|link=Team:DTU-Denmark/Project_experiment|Chromosomal knockout]]
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Experiments were performed to verify that the envisioned small RNA based gene silencing is possible. Plasmids containing and strains deleted for the components were constructed providing a biological model.
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[[Team:DTU-Denmark/Project_experiment|Read more...]]
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== Bioinformatics ==
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[[File:DTU1 Sequence logo.png|200px|frameless|right|link=Team:DTU-Denmark/Bioinformatic]]
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A bioinformatics study was performed to investigate the possibilities of engineering the trap-RNA system to target any gene. The study elucidates interesting features of sequence and secondary structure conservation guiding future application.
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[[Team:DTU-Denmark/Bioinformatic|Read more...]]
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== Modeling ==
== Modeling ==
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[[File:DTU2011_modeling_fig.png|200px|thumb|right|'''Kinetic models''' of the system are the basis for modeling. Blue is target mRNA, red is small RNA and green is trap-RNA]]
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[[File:DTU2011_modeling_fig.png|200px|frameless|right|link=Team:DTU-Denmark/Modeling|'''Kinetic models''' of the system are the basis for modeling. Blue is target mRNA, red is small RNA and green is trap-RNA]]
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A framework for characterization of gene silencing was developed to guide rational design and test hypotheses. Steady state analysis revealed that each trap-RNA system has a characteristic fold repression.
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[[Team:DTU-Denmark/Modeling|Read more...]]
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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 [[Team:DTU-Denmark/Modeling|modeling]].
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== Bioinformatic ==
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== Experiment: Improving araBAD ==
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[[File:DTU-Relative_promoter_activity.png|200px|right|link=Team:DTU-Denmark/Project_improving_araBAD]]
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The dynamic range of the araBAD promoter was expanded by either changing the -10 and -35 elements or randomly changing the promoter using a synthetic promoter library. Experiments were performed characterizing the constructed promoters.
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[[Team:DTU-Denmark/Project_improving_araBAD|Read more..]]
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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 [[Team:DTU-Denmark/Bioinformatic|bioinformatic]].
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== Data page ==
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[[File:DTU1_Data_page-figure1.png|200px|right|link=Team:DTU-Denmark/Data]]
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The data page provides a description of the constructed BioBricks and how they work. It furthermore provides full links to the iGEM parts registry enabling easy retrieval of each submitted part.
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[[Team:DTU-Denmark/Data|Read more...]]
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==References==
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[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).
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[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.  
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== Results ==
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The '''bioinformatic study''' revealed some interesting constrains for engineering novel sRNAs with our gene silencing tool; a terminal poly-U tail, a putative Hfq binding site, a stemloop without sequence constrains, and a terminal stemloop with high sequence conservation. See the results [[Team:DTU-Denmark/Bioinformatic#Results|here]].
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[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).
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'''Testing sRNA''' we were able to show that the target gene chiP can be placed on a plasmid and be regulated by a small RNA. Changing the complementary sequence removes this regulation. See the results [[Team:DTU-Denmark/Project_testing_sRNA#Results_and_Conclusions|here]].
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[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).
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Through rational design of the '''araBAD promoter''' we managed to reach a higher level of expression from that promoter. Also, we proved that Synthetic Promoter Library can be created for an inducible promoter to expand its dynamic range. See the results [[Team:DTU-Denmark/Project_improving_araBAD#Results|here]].
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[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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The '''modeling''' provides a guideline for [[Team:DTU-Denmark/Modeling#Parameters_and_proposed_experiments|determining parameters]] and design of [[Team:DTU-Denmark/Modeling#Design_of_dynamic_range|dynamic range]] of our gene silencing tool. A simulation revealed fast dynamics of gene silencing. See the results [[Team:DTU-Denmark/Modeling#Dynamics|here]].
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Latest revision as of 03:41, 22 September 2011

Overview

Abstract

Small regulatory RNA is an active area of research with untapped possibilities for application in biotechnology. Such applications include convenient gene silencing and fine-tuning of gene expression, which are currently cumbersome processes restricted to well studied bacteria. We have investigated a novel type of RNA regulation based on the chitobiose system, where the inhibition caused by a small RNA is relieved by another small RNA called trap-RNA.
Two-level sRNA regulation. Blue is any target mRNA, red is sRNA and green is trap-RNA.
We explore the possibility of using the system to uniquely target and repress any gene of interest, potentially providing unprecedented specificity and control of gene silencing. We furthermore constructed araBAD promoters with varying promoter activities using synthetic promoter libraries.

Experiment: Testing sRNA

Chromosomal knockout

Experiments were performed to verify that the envisioned small RNA based gene silencing is possible. Plasmids containing and strains deleted for the components were constructed providing a biological model. Read more...

Bioinformatics

DTU1 Sequence logo.png

A bioinformatics study was performed to investigate the possibilities of engineering the trap-RNA system to target any gene. The study elucidates interesting features of sequence and secondary structure conservation guiding future application. Read more...

Modeling

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

A framework for characterization of gene silencing was developed to guide rational design and test hypotheses. Steady state analysis revealed that each trap-RNA system has a characteristic fold repression. Read more...

Experiment: Improving araBAD

DTU-Relative promoter activity.png

The dynamic range of the araBAD promoter was expanded by either changing the -10 and -35 elements or randomly changing the promoter using a synthetic promoter library. Experiments were performed characterizing the constructed promoters. Read more..

Data page

DTU1 Data page-figure1.png

The data page provides a description of the constructed BioBricks and how they work. It furthermore provides full links to the iGEM parts registry enabling easy retrieval of each submitted part. Read more...

Results

The bioinformatic study revealed some interesting constrains for engineering novel sRNAs with our gene silencing tool; a terminal poly-U tail, a putative Hfq binding site, a stemloop without sequence constrains, and a terminal stemloop with high sequence conservation. See the results here.

Testing sRNA we were able to show that the target gene chiP can be placed on a plasmid and be regulated by a small RNA. Changing the complementary sequence removes this regulation. See the results here.

Through rational design of the araBAD promoter we managed to reach a higher level of expression from that promoter. Also, we proved that Synthetic Promoter Library can be created for an inducible promoter to expand its dynamic range. See the results here.

The modeling provides a guideline for determining parameters and design of dynamic range of our gene silencing tool. A simulation revealed fast dynamics of gene silencing. See the results here.

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