Difference between revisions of "Part:BBa K2406051"
 
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<partinfo>BBa_K2406051 short</partinfo>
 
<partinfo>BBa_K2406051 short</partinfo>
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==Introduction==
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This measurement construct was used to test the self-reactivity of Rox <partinfo>BBa_K2406000</partinfo>. The theory behind the function of this measurement construct is summarised in the adjacent figure. Essentially, when two recombination sites cannot be recognised by a single recombinase, the terminator (represented as parallel lines in the diagram) will not be excised and there will be no RFP reporter outlook. This part is useful because it tests the self-reactivity of Rox, verifying that our recombinase generator <partinfo>BBa_K2406081</partinfo> and Rox target site <partinfo>BBa_K2406000</partinfo> function as a recombinase/target site system.  Thus, this measurement construct demonstrates that Dre/Rox functions in E. coli
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[[File:Edinburgh UG measurement constructs.png |200px|thumb|left| Schematic outlining principle of all measurement constructs used by Edinburgh_UG 2017]]
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==Results==
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All assays performed using this measurement construct are summarised to the right. For reference, cross-reactivity and fluorescence output is compared to other measurement constructs in the context of Dre recombinase<partinfo>BBa_K2406081</partinfo> and Rox target sites <partinfo>BBa_K2406000</partinfo>.
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We observed expected reactivity within this construct, as fluorescence output was observed when the Dre recombinase was present, as induced by a pulse of IPTG.
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[[File:Dre measurements.png |200px|thumb|left|All assays performed involving Dre recombinase]]
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[[File:Rox Assays.png |200px|thumb|left|All assays performed involving Rox target sites]]
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==Discussion==
 +
The target sites involved in this construct were previously identified as working in a site-specific manner [1]. This test verifies their function as biobrick parts used in E. coli. Our results show that the measurement construct works as expected, confirming the activity of Dre recombinase<partinfo>BBa_K2406081</partinfo> and its associated target site Rox <partinfo>BBa_K2406000</partinfo>. Therefore, this measurement construct confirmed the activity of these parts and demonstrated that they are suitable for carrying out a site-specific reaction within cells.
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==References==
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[1]Anastassiadis, K., Fu, J., Patsch, C., Hu, S., Weidlich, S., Duerschke, K., Buchholz, F., Edenhofer, F., and Stewart A.F. 2009. “Dre recombinase, like Cre, is a highly efficient site-specific recombinase in E. coli, mammalian cells and mice.” Disease Models and Mechanisms: Sep-Oct; 2(9-10):508-515.
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==Sequences==
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File below confirms sequence of all target sites, generators and measurement constructs used.
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[[Media:File:Sequencing Results Edinburgh UG.zip]]
  
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Rox-Term-Rox construct inserted in BBa_J04450 part. If Dre recombinase is present it will cause recombination between the two rox sites, excising the terminator and allowing expression of the RFP protein. Therefore, this plasmid can be used as an assay for determining levels of Dre recombinase activity.
 
  
 
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===Usage and Biology===
 
===Usage and Biology===
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Recombinases are crucial tools for biotechnology. As our designed proofs of concept illustrate, they are excellent tools for dynamic manipulation of gene expression. To accurately test for recombinase function, a simple, reliable assay is required. This is provided by our target site-term-target site constructs. These are inserted between a promoter and an RBS for a fluorescent protein. If the recombinase recognises the target sites, then recombination will occur and the terminator will be excised, leading to expression of the fluorescent reporter. This construct was used to assay Dre/Rox recombination activity and efficiency.  
 
Recombinases are crucial tools for biotechnology. As our designed proofs of concept illustrate, they are excellent tools for dynamic manipulation of gene expression. To accurately test for recombinase function, a simple, reliable assay is required. This is provided by our target site-term-target site constructs. These are inserted between a promoter and an RBS for a fluorescent protein. If the recombinase recognises the target sites, then recombination will occur and the terminator will be excised, leading to expression of the fluorescent reporter. This construct was used to assay Dre/Rox recombination activity and efficiency.  
 
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Latest revision as of 10:38, 29 October 2017


Rox-Term-Rox Measurement construct.

Introduction

This measurement construct was used to test the self-reactivity of Rox BBa_K2406000. The theory behind the function of this measurement construct is summarised in the adjacent figure. Essentially, when two recombination sites cannot be recognised by a single recombinase, the terminator (represented as parallel lines in the diagram) will not be excised and there will be no RFP reporter outlook. This part is useful because it tests the self-reactivity of Rox, verifying that our recombinase generator BBa_K2406081 and Rox target site BBa_K2406000 function as a recombinase/target site system. Thus, this measurement construct demonstrates that Dre/Rox functions in E. coli

Schematic outlining principle of all measurement constructs used by Edinburgh_UG 2017

Results

All assays performed using this measurement construct are summarised to the right. For reference, cross-reactivity and fluorescence output is compared to other measurement constructs in the context of Dre recombinaseBBa_K2406081 and Rox target sites BBa_K2406000. We observed expected reactivity within this construct, as fluorescence output was observed when the Dre recombinase was present, as induced by a pulse of IPTG.

All assays performed involving Dre recombinase
All assays performed involving Rox target sites

Discussion

The target sites involved in this construct were previously identified as working in a site-specific manner [1]. This test verifies their function as biobrick parts used in E. coli. Our results show that the measurement construct works as expected, confirming the activity of Dre recombinaseBBa_K2406081 and its associated target site Rox BBa_K2406000. Therefore, this measurement construct confirmed the activity of these parts and demonstrated that they are suitable for carrying out a site-specific reaction within cells.

References

[1]Anastassiadis, K., Fu, J., Patsch, C., Hu, S., Weidlich, S., Duerschke, K., Buchholz, F., Edenhofer, F., and Stewart A.F. 2009. “Dre recombinase, like Cre, is a highly efficient site-specific recombinase in E. coli, mammalian cells and mice.” Disease Models and Mechanisms: Sep-Oct; 2(9-10):508-515.

Sequences

File below confirms sequence of all target sites, generators and measurement constructs used. Media:File:Sequencing Results Edinburgh UG.zip


Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal AgeI site found at 876
    Illegal AgeI site found at 988
  • 1000
    COMPATIBLE WITH RFC[1000]