Difference between revisions of "Part:BBa K494001"

(New page: In general we want to provide a new principle of gene regulation which can be further developed, tested and optimizted by everybody. Therefore we focus on providing the parts needed for ve...)
 
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In general we want to provide a new principle of gene regulation which can be further developed, tested and optimizted by everybody. Therefore we focus on providing the parts needed for verification and testing of new individual switches. We provide a plasmid which can be used for further cloning, a positive control to test the general functionality and the constructs we characterized for comparison.
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In general we want to provide a new, scalable principle of gene regulation based on termination and antitermination which can be further developed, tested and optimizted by everybody. To enable that in the easiest way possible we decided to not just add the switches we designed but instead the material needed for evaluation. Therefore we focus on providing the parts needed for verification and testing of new individual switches. <br><br>
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[edit] Screening system: Backbone BBa_K494001
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We designed a new screening systems based on the non-functional pSB1A10 plasmid. We improved its features for ‘’in vivo’’ characterization of PoPS-based devices using fluorescent reporters . The plasmid still contains the Pbad arabinose-inducible induction system as a tunable input and eGFP as an internal standard for induction. However, we altered the reporter protein to mCherry. Furthermore we adjusted the BioBrick cloning site to allow cloning of additional parts independent from the Input/Output measurement. This screening plasmid is designed to be used with a second Arabinose inducible promoter BBa_I13453 which is not included in this part.
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With this BioBrick we provide a backbone which allows further cloning to test individual switches which can be easily designed using the principles described in the [http://2010.igem.org/Team:TU_Munich/Project TU Munich iGEM 2010 wiki].  
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The improved screening system is optimized for the evaluation of PoPS-based devices in fluorescence measurements. RFP which was known to contain an RNase restriction site was replaced by mCherry which combines good expression yield, short maturation times and an acceptable and well-characterized quantum yield. A unique challenge for the characterization of our switches is the expression of a corresponding signal independent of the input/output measurement. Thus, we moved the BioBrick cloning site resulting in the fluorescent reporter being inside the cloning site and giving the possibility to clone independent parts behind the reporter protein. To fully function our screening plasmid need the arabinose inducible promoter BBa_I13453 in front of the PoPS-based device to screen. Using a second arabinose inducible promoter, we were able to keep eGFP as an internal standard for the tunable input via the Pbad arabinose-inducible induction system. The two identical promoters ensure the same rate of induction for eGFP and the tested PoPS-based device. Thus, obtaining comparable screening results is easy. Unfortunately, this design implicates a minor disadvantage. Two cloning steps are needed to gain an functional construct for testing any PoPS-based device.
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The improved screening system, based on the pSB1A10 plasmid, is optimized for the evaluation of PoPS-based devices in fluorescence measurements. RFP which was known to contain an RNase restriction site was replaced by mCherry which combines good expression yield, short maturation times and an acceptable and well-characterized quantum yield. A unique challenge for the characterization of our switches is the expression of a corresponding signal independent of the input/output measurement. Thus, we moved the BioBrick cloning site resulting in the fluorescent reporter being inside the cloning site and giving the possibility to clone independent parts behind the reporter protein. To fully function our screening plasmid need the arabinose inducible promoter BBa_I13453 in front of the PoPS-based device to screen. Using a second arabinose inducible promoter, we were able to keep eGFP as an internal standard for the tunable input via the Pbad arabinose-inducible induction system. The two identical promoters ensure the same rate of induction for eGFP and the tested PoPS-based device. Thus, obtaining comparable screening results is easy. Unfortunately, this design implicates a minor disadvantage: Two cloning steps are needed to gain an functional construct for testing any PoPS-based device.
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For exemplary usage visit BBa_K494003 - BBa_K494006. A positive control for mCherry expression can be found in BBa_K494002.

Revision as of 09:46, 27 October 2010

In general we want to provide a new, scalable principle of gene regulation based on termination and antitermination which can be further developed, tested and optimizted by everybody. To enable that in the easiest way possible we decided to not just add the switches we designed but instead the material needed for evaluation. Therefore we focus on providing the parts needed for verification and testing of new individual switches.

With this BioBrick we provide a backbone which allows further cloning to test individual switches which can be easily designed using the principles described in the [http://2010.igem.org/Team:TU_Munich/Project TU Munich iGEM 2010 wiki].

The improved screening system, based on the pSB1A10 plasmid, is optimized for the evaluation of PoPS-based devices in fluorescence measurements. RFP which was known to contain an RNase restriction site was replaced by mCherry which combines good expression yield, short maturation times and an acceptable and well-characterized quantum yield. A unique challenge for the characterization of our switches is the expression of a corresponding signal independent of the input/output measurement. Thus, we moved the BioBrick cloning site resulting in the fluorescent reporter being inside the cloning site and giving the possibility to clone independent parts behind the reporter protein. To fully function our screening plasmid need the arabinose inducible promoter BBa_I13453 in front of the PoPS-based device to screen. Using a second arabinose inducible promoter, we were able to keep eGFP as an internal standard for the tunable input via the Pbad arabinose-inducible induction system. The two identical promoters ensure the same rate of induction for eGFP and the tested PoPS-based device. Thus, obtaining comparable screening results is easy. Unfortunately, this design implicates a minor disadvantage: Two cloning steps are needed to gain an functional construct for testing any PoPS-based device.

For exemplary usage visit BBa_K494003 - BBa_K494006. A positive control for mCherry expression can be found in BBa_K494002.