Difference between revisions of "Part:BBa K2406063"

 
 
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<partinfo>BBa_K2406063 short</partinfo>
 
<partinfo>BBa_K2406063 short</partinfo>
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==Introduction==
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This part was designed to test if Slox and Vlox Target sites were orthogonal. If either SCre 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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This measurement construct was used to test the cross-reactivity of Slox and Vlox (<partinfo>BBa_K2406003</partinfo>, <partinfo>BBa_K2406002</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 SCre/Slox 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 SCre and VCre recombinases (<partinfo>BBa_K2406084</partinfo>, <partinfo>BBa_K2406083</partinfo>). We observed unexpected cross-reactivity within this construct, as shown by relatively highfluorescence output when SCre and VCre recombinases (<partinfo>BBa_K2406084</partinfo>, <partinfo>BBa_K2406083</partinfo>) were present.
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[[File:SCre Assays.png |200px|thumb|left|All assays performed involving VCre]]
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[[File:VCre Assays.png|200px|thumb|left|All assays performed involving SCre]]
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==Discussion==
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The target sites involved in this construct were previously claimed to be orthogonal to one another [1]. However, our cross-reactivity tests in E. coli contradict the existing literature. Unexpectedly, we observed cross-reactivity between these two target sites <partinfo>BBa_K2406003</partinfo>, <partinfo>BBa_K2406002</partinfo>. This runs counter to the originally reported orthogonality between these systems [1]. Therefore, this indicates that VCre/Vlox and SCre/Slox cannot be used to catalyse distinct and independent recombination events in one cell.
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==References==
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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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Latest revision as of 12:00, 29 October 2017


Slox-Term-Vlox Measurement Construct

Introduction

This measurement construct was used to test the cross-reactivity of Slox and Vlox (BBa_K2406003, BBa_K2406002). 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 SCre/Slox 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 SCre and VCre recombinases (BBa_K2406084, BBa_K2406083). We observed unexpected cross-reactivity within this construct, as shown by relatively highfluorescence output when SCre and VCre recombinases (BBa_K2406084, BBa_K2406083) were present.

All assays performed involving VCre
All assays performed involving SCre

Discussion

The target sites involved in this construct were previously claimed to be orthogonal to one another [1]. However, our cross-reactivity tests in E. coli contradict the existing literature. Unexpectedly, we observed cross-reactivity between these two target sites BBa_K2406003, BBa_K2406002. This runs counter to the originally reported orthogonality between these systems [1]. Therefore, this indicates that VCre/Vlox and SCre/Slox cannot be used to catalyse distinct and independent recombination events in one cell.

References

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 888
    Illegal AgeI site found at 1000
  • 1000
    COMPATIBLE WITH RFC[1000]