Team:DTU-Denmark/Project

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{{:Team:DTU-Denmark/Templates/Standard_page_begin|Project}}
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{{:Team:DTU-Denmark/Templates/Standard_page_begin|Overview}}
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__NOTOC__
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<div class="overviewPage">
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== Tuning regulation with a non-coding RNA trap ==
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<div class="overviewBox right">
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[[File:DTU2011_project_fig1.png|200px|thumb|right|caption]]
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Small regulatory RNA is an active area of research with untapped possibilities for application in biotechnology. One such application could be the optimization and fine-tuning of synthetic biological circuits, which is currently a cumbersome process of trial and error. We have investigated a novel type of RNA regulation<span class="superscript">[[#References|[2]]]</span><span class="superscript">[[#References|[5]]]</span>, where the inhibition caused by a small regulatory RNA is relieved by another RNA called trap-RNA. The system displays a large dynamic range and can uniquely target and repress any gene of interest providing unprecedented flexibility. We suspect that any level of repression is achievable by simply altering the sequences of the involved RNAs. Multiple such systems can coexist without interfering and are thus compatible with more complex designs. Furthermore the trap-RNA can be fused to any transcript in effect allowing any gene to act as an activator.
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== Overview ==
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Our project is a proof of concept project, showing that the ''E. coli'' sRNA ''sroB'' and the intergenic sRNA in the ''chbBCARG'' gene can be used to control gene expression by targeting the ''ybfM'' Shine-Delgarno, and that these sRNAs can be rationally designed to target other Shine-Delgarnos.
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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 experimental part of the project can be broken into 3 distinct parts, which combined form the complete project:
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* Construction of plasmids
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<div class="overviewBox left">
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* Strain construction
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* Improving the ''araBAD'' promoter
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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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== 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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'''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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</div>
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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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== Plasmids ==
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<div class="overviewBox right">
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There are three elements in the natural trap RNA system that regulate chitobiose metabolism in E. coli: (1) chitoporin ''chiP'' (alias ''ybfM'') that facilitates uptake of chitosugars from the medium into the cell; (2) sRNA ''chiX'' (alias ''sroB'', ''micM'') which post-transcriptionally regulates ''chiP''; and (3) ''chiXR'' sRNA which is transcribed from intergenic region in ''chbBCARG'' operon and regulates ''chiX'' sRNA. ChiX sRNA regulates chiP expression through binding to the Shine-Dalgarno sequence on the ''chiP'' mRNA and thus inhibits recruitment of 30S ribosome subunit and translation cannot occur. Only when chitobiose is present in the medium can the ''chiXR'' be transcribed and subsequently its transcript can bind to ''chiX'' sRNA, releasing ''chiP'' repression.
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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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In order to quantitatively describe the natural trap RNA system following three plasmids were constructed:
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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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*PchiP-lacZ plasmid
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</div>
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: To mimic and test repression of the chiP gene its upstream region of 599 bp was fused to reporter genes lacZ and GFP and inserted on the pSLD39 plasmid. This constructs were controlled by constitutive promoter. LacZ codes for $\beta$-galactosidase and its activity was tested using $\beta$-Gal assay while GFP activity was tested using Biolektor.
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*Ptet-chiX plasmid
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<div class="overviewBox left">
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: ChiX, which is 84 bp long, was inserted into pSB4K plasmid together with regulatory element from ''tet'' operon. Expression from Ptet promoter was induced using anhydrotetracycline.
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*PBAD-chiXR plasmid
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== Bioinformatics ==
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: Introgenic region from chbBCARG operon 284 bp long was inserted into pBAD18 plasmid. Expression from that plasmid was controlled by regulatory element from ''ara'' operon with arabinose as inducer.
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[[File:DTU1 Sequence logo.png|200px|frameless|right|link=Team:DTU-Denmark/Bioinformatic]]
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[[File:DTU1_Plasmids.png|665px|thumb|center|add some text]]
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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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== Strain ==
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</div>
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The strain used in testing our system is based on the ''E. coli'' strain W3110, a wild-type strain of ''Escherichia coli'' K-12<span class="superscript">[[#References|[3]]]</span> with the ''lacZYA'', ''chbBCARG'' operons and ''chiP'' (alias ''ybfM''), ''chiX'' (alias ''sroB'', ''micM'') genes deleted from the chromosome. The three latter code for utilization of chitobiose (a sugar), chitobiose permease and the regulation of the chitobiose permease respectively.
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A procedure known as redswap<span class="superscript">[[#References|[1]]]</span> was used to delete these genes. Schematically the procedure works in the following manner:
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<div class="overviewBox right">
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#Transform competent cells with Red and cre helper plasmids.
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== Modeling ==
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#Make the cells competent and induce the Red recombinase.
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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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#Transform with linear piece of DNA.
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#Select for gain of resistance.
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#Move to medium inducing the Cre recombinase.
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#Screen for loss of resistance.
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#Verify the construct.
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#Repeat step 2-7 as needed.
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#Cure the helper plasmids.
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The Red helper plasmids encodes the $\lambda{}$ phage Red recombination system. This system allows recombination events between sequences with around 40 nucleotides of homology.
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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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The cre helper plasmid encodes the Cre recombinase. This recombinase recognises 34bp ''loxP'' sequences and promotes recombination between them.
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[[Team:DTU-Denmark/Modeling|Read more...]]
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An example of a linear piece of DNA that could be used in redswap, as well as the simplified representation of the procedure, can be seen in figure \ref{linDNA}.
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[[File:DTU1_Redswap.png|665px|thumb|center|add some text]]
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<div class="overviewBox left">
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[[File:DTU1_Lox_sites.png|400px|thumb|right|add some text]]
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== Experiment: Improving araBAD ==
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In case of two or more subsequent gene deletions using redswap a problem of recombination between an old ''loxP'' site and newly introduced ones arises. Remember, that after each round of redswap one ''loxP'' site is left in the genome. As a result recombination using Cre recombinase in second or next redswap might not occur between loxP sites flanking a resistance gene. To circumvent this problem two mutated loxP sequences are used - ''lox66'' and ''lox71''<span class="superscript">[[#References|[4]]]</span>. In spite of the mutations these lox sites are recognized by Cre which can remove the intervening region. Finally, from lox66 and lox71 a new site is formed, ''lox72'', which has a reduced affinity to Cre recombinase. Thus, more than one gene deletions can be subsequently conducted.
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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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</div>
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<div style="clear: both;"></div>
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<div class="overviewBox right">
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<html></div><div class="whitebox article"></html>
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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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</div>
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==References==
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<div class="overviewBox left">
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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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</div>
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<div style="clear: both;"></div>
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</div>
{{:Team:DTU-Denmark/Templates/Standard_page_end}}
{{:Team:DTU-Denmark/Templates/Standard_page_end}}

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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