Difference between revisions of "Part:BBa K2406065"

 
 
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<partinfo>BBa_K2406065 short</partinfo>
 
<partinfo>BBa_K2406065 short</partinfo>
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==Introduction==
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Slox-Term-Slox construct inserted in BBa_J04450 part. If SCre recombinase is present it will cause recombination between the two Slox sites, excising the terminator and allowing expression of the RFP protein. Therefore, this plasmid can be used as an assay for determining levels of SCre recombinase activity.
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This measurement construct was used to test the self-reactivity of Slox <partinfo>BBa_K2406003</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 Slox, verifying that our recombinase generator <partinfo>BBa_K2406084</partinfo> and Slox target site <partinfo>BBa_K2406003</partinfo> function as a recombinase/target site system.  Thus, this measurement construct demonstrates that SCre/Slox 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 SCre recombinase<partinfo>BBa_K2406084</partinfo> and Slox target sites <partinfo>BBa_K2406003</partinfo>.
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We observed expected reactivity within this construct, as fluorescence output was observed when the SCre recombinase was present, as induced by a pulse of IPTG.
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[[File:SCre Assays.png |200px|thumb|left|All assays performed involving SCre recombinase]]
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[[File:Slox Assays.png |200px|thumb|left|All assays performed involving Slox Assays.png 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 SCre recombinase<partinfo>BBa_K2406084</partinfo> and its associated target site Slox <partinfo>BBa_K2406003</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]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]]
  
  

Latest revision as of 10:57, 29 October 2017


Slox-Term-Slox Measurement Construct

Introduction

This measurement construct was used to test the self-reactivity of Slox BBa_K2406003. 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 Slox, verifying that our recombinase generator BBa_K2406084 and Slox target site BBa_K2406003 function as a recombinase/target site system. Thus, this measurement construct demonstrates that SCre/Slox 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 SCre recombinaseBBa_K2406084 and Slox target sites BBa_K2406003. We observed expected reactivity within this construct, as fluorescence output was observed when the SCre recombinase was present, as induced by a pulse of IPTG.

All assays performed involving SCre recombinase
All assays performed involving Slox Assays.png 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 SCre recombinaseBBa_K2406084 and its associated target site Slox BBa_K2406003. 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]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]