Difference between revisions of "Part:BBa K2406053"

 
 
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<partinfo>BBa_K2406053 short</partinfo>
 
<partinfo>BBa_K2406053 short</partinfo>
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==Introduction==
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Vox-Term-Vox construct inserted in BBa_J04450 part. If Vika recombinase is present it will cause recombination between the two Vox sites, excising the terminator and allowing expression of the RFP protein. Therefore, this plasmid can be used as an assay for determining levels of Vika recombinase activity.
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This measurement construct was used to test the self-reactivity of Vox <partinfo>BBa_K2406001</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 Vox, verifying that our Vika recombinase generator <partinfo>BBa_K2406081</partinfo> and Vox target site <partinfo>BBa_K2406001</partinfo> function as a recombinase/target site system.  Thus, this measurement construct demonstrates that Vika/vox 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 Vika recombinase<partinfo>BBa_K2406082</partinfo> and Vox target sites <partinfo>BBa_K2406001</partinfo>.
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We observed expected reactivity within this construct, as fluorescence output was observed when the Vikarecombinase was present, as induced by a pulse of IPTG.
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[[File:Vika_assays.png |200px|thumb|left|All assays performed involving Vika recombinase]]
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[[File:Vox Assays.png |200px|thumb|left|All assays performed involving Vox target sites]]
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==Discussion==
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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 Vika recombinase<partinfo>BBa_K2406082</partinfo> and its associated target site Vox <partinfo>BBa_K2406001</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]Karimova, M., Abi-Ghanem, J., Berger, N., Surendranath, V., Pisabarro, M.T., Buchholz, F. 2013 “Vika/vox, a novel efficient and specific Cre/loxP-like site-specific recombination system”. Nucleic Acids Research 41(2):e37.
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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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===Usage and Biology===
 
===Usage and Biology===
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The main goal of our project in the short-term was to develop and test recombinase parts in E. coli. Most of the proteins had not previously been tested in bacteria before. Therefore, the new parts we have developed and tested are recombinases and their target sites. We then confirmed the activity of the recombinase proteins and function of target sites with the composite parts described below. In addition, we developed an improved promoter part. It will allow for tightly-regulated expression of our recombinases in both prokaryotes and eukaryotes. This construct in particular assayed Vika/Vox recombination
  
 
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Latest revision as of 10:47, 29 October 2017


Vox-Term-Vox Measurement Construct

Introduction

This measurement construct was used to test the self-reactivity of Vox BBa_K2406001. 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 Vox, verifying that our Vika recombinase generator BBa_K2406081 and Vox target site BBa_K2406001 function as a recombinase/target site system. Thus, this measurement construct demonstrates that Vika/vox 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 Vika recombinaseBBa_K2406082 and Vox target sites BBa_K2406001. We observed expected reactivity within this construct, as fluorescence output was observed when the Vikarecombinase was present, as induced by a pulse of IPTG.

All assays performed involving Vika recombinase
All assays performed involving Vox 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 Vika recombinaseBBa_K2406082 and its associated target site Vox BBa_K2406001. 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]Karimova, M., Abi-Ghanem, J., Berger, N., Surendranath, V., Pisabarro, M.T., Buchholz, F. 2013 “Vika/vox, a novel efficient and specific Cre/loxP-like site-specific recombination system”. Nucleic Acids Research 41(2):e37.

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 880
    Illegal AgeI site found at 992
  • 1000
    COMPATIBLE WITH RFC[1000]