Team:Heidelberg/Notebook/BSDesign

From 2010.igem.org

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==Introduction==
==Introduction==
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Actual ideas for therapeutic treatments of diseases like (???) include the use of virus for cell or tissue specific "drug" targeting. Where the delivery by the virus may cure the patient if targeted to the right cell, major problems are side effects coming up when mistargeted virus deliver their cargo to non-targeted cells. As the standard load of a virus is DNA and the pharmacological function results in expression of its information, our idea is to control the delivered DNA outcome. Therefore we want to use cell type specific micro RNA (miRNA) expression levels for activation of the therapeutic treatment.
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To create binding site (BS) patterns for micro RNAs (miRNAs) , we used the random assembly PCR (raPCR) – method from iGEM2009-Heidelberg team ([https://2009.igem.org/Team:Heidelberg/Project_Synthetic_promoters#RA-PCR_protocol see here]) and adopted it to our purposes.
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The differences:
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*Sequences from 100 to 400 base pairs are requested.
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*Oligos span over a whole binding site for a certain miRNA and shuffling occurs on the level of pattern creation.
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As miRNAs are naturally used in cells to regulate translation, usage of the existing system for self-regulation sounds obvious. So we tried to create binding sites for miRNAs to target our constructs and either shut-down (off-targeting) or activate (on-targeting) gene expression in targeted tissues. In the off-targeting strategy the therapeutic gene is itself targeted for RNAi, so expression will persist only in non-targeted cells. In the on-targeting strategy, two different constructs built up an operator/repressor system (e.g. Tet-On/Off), where the regulator of the therapeutic gene, the repressor, is targeted for RNAi. Only cells which are able to suppress translation of the repressor will be affected from the therapeutic gene.
 
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To achieve this, we used the random assembly PCR (raPCR) – method from iGEM2009-Heidelberg team and adopted it to our purposes.
 
-
The differences: Sequences from 100 to 400 base pairs are requested. Oligos span over a whole binding site for a certain miRNA and shuffling occurs on the level of pattern creation.
 
Several points need to be considered for setting up miRNA-binding site (miRBS) patterns:
Several points need to be considered for setting up miRNA-binding site (miRBS) patterns:
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:*the right distance after the stop codon for efficient (or non-efficient) targeting of miRNAs
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:*the right distance after the stop codon for efficient (or non-efficient) BS recognition
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:*distance and sequence between miRBS (spacer region)
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:*distance and sequence between miRBS (the spacer)
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See on our Notebook pages how we created binding site patterns.
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The adopted method for BS-patterns can be found on our [https://2010.igem.org/Team:Heidelberg/Notebook/Methods#random_assembly_PCR methods page].
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On the [synthetic_microRNA_binding_Site_patterns_against_endogenous_miRNA Parts]-Page you can find standardized BS-patterns for hsa-mir-122 and has-mir-221, containing at least 2 binding sites.
{{:Team:Heidelberg/Single_Bottom}}
{{:Team:Heidelberg/Single_Bottom}}

Revision as of 22:29, 27 October 2010

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Binding Site Design

Introduction

To create binding site (BS) patterns for micro RNAs (miRNAs) , we used the random assembly PCR (raPCR) – method from iGEM2009-Heidelberg team (see here) and adopted it to our purposes. The differences:

  • Sequences from 100 to 400 base pairs are requested.
  • Oligos span over a whole binding site for a certain miRNA and shuffling occurs on the level of pattern creation.

Several points need to be considered for setting up miRNA-binding site (miRBS) patterns:

  • the right distance after the stop codon for efficient (or non-efficient) BS recognition
  • distance and sequence between miRBS (the spacer)


See on our Notebook pages how we created binding site patterns.

The adopted method for BS-patterns can be found on our methods page.

On the [synthetic_microRNA_binding_Site_patterns_against_endogenous_miRNA Parts]-Page you can find standardized BS-patterns for hsa-mir-122 and has-mir-221, containing at least 2 binding sites.


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