Difference between revisions of "Part:BBa K2406057"

 
 
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__NOTOC__
 
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<partinfo>BBa_K2406057 short</partinfo>
 
<partinfo>BBa_K2406057 short</partinfo>
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==Introduction==
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This measurement construct was used to test the cross-reactivity of Vlox and LoxP (<partinfo>BBa_K2406002</partinfo>, <partinfo>BBa_K1680005</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 cross-reactivity of the target sites in question. In order to catalyse two independent, distinct recombination events in one cell with two recombinase systems, it is vital that there is no cross-reactivity. Thus, this measurement construct tests the suitability for using Cre/Lox and VCre/Vlox in one cell.
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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 VCre <partinfo>BBa_K2406083</partinfo> and Vlox <partinfo>BBa_K2406002</partinfo>.
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We observed no cross-reactivity within this construct, as fluorescence output was negligible when VCre was present and not present. 
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[[File:VCre Assays.png |200px|thumb|left|All measurements performed involving VCre]]
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[[File:Vlox Assays.png|200px|thumb|left|All measurements performed involving Vlox target sites]]
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==Discussion==
 +
The target sites involved in this construct were previously discovered as being potentially orthogonal to other recombinases [1]. It is therefore important to test their cross-reactivity extensively to fully understand what recombinases can be used within one cell. This had not been done extensively before. Our results demonstrate no cross reactivity between the two target sites, <partinfo>BBa_K2406002</partinfo>, <partinfo>BBa_K1680005</partinfo>. This is because negligible RFP output was seen compared to control. Therefore, the VCre/Vlox and Cre/LoxP can be used in an orthogonal manner within a single cell.
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==References==
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[1]Suzuki E. and Nakayama, M. 2011. “VCre/VloxP and SCre/SloxP: new site-specific recombination systems for genome engineering.” Nucleic Acids Research 39(8):e49.
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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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This part was designed to test if Vlox and Lox Target sites were orthogonal. If either Cre or VCre recombinase could recognise both sites, it would catalyse recombination leading to expression of RFP. If the target sites were orthogonal, no RFP would be produced.
 
  
 
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Latest revision as of 11:11, 29 October 2017


Vlox-Term-Lox Measurement Construct

Introduction

This measurement construct was used to test the cross-reactivity of Vlox and LoxP (BBa_K2406002, BBa_K1680005). 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 cross-reactivity of the target sites in question. In order to catalyse two independent, distinct recombination events in one cell with two recombinase systems, it is vital that there is no cross-reactivity. Thus, this measurement construct tests the suitability for using Cre/Lox and VCre/Vlox in one cell.

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 VCre BBa_K2406083 and Vlox BBa_K2406002. We observed no cross-reactivity within this construct, as fluorescence output was negligible when VCre was present and not present.

All measurements performed involving VCre
All measurements performed involving Vlox target sites

Discussion

The target sites involved in this construct were previously discovered as being potentially orthogonal to other recombinases [1]. It is therefore important to test their cross-reactivity extensively to fully understand what recombinases can be used within one cell. This had not been done extensively before. Our results demonstrate no cross reactivity between the two target sites, BBa_K2406002, BBa_K1680005. This is because negligible RFP output was seen compared to control. Therefore, the VCre/Vlox and Cre/LoxP can be used in an orthogonal manner within a single cell.

References

[1]Suzuki E. and Nakayama, M. 2011. “VCre/VloxP and SCre/SloxP: new site-specific recombination systems for genome engineering.” Nucleic Acids Research 39(8):e49.

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 912
    Illegal AgeI site found at 1024
  • 1000
    COMPATIBLE WITH RFC[1000]