Difference between revisions of "Part:BBa K2406000"

 
 
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<partinfo>BBa_K2406000 short</partinfo>
 
<partinfo>BBa_K2406000 short</partinfo>
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==Introduction==
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Dre Recombinase <partinfo>BBa_K2406081</partinfo> is a tyrosine recombinase that catalyses recombination between Rox target sites [1]. This can lead to integration, excision, or inversion of the DNA sequence in between these target sites. This is an example of a site-specific recombinase (SSR). SSRs have long been recognised to be excellent biological tools, used in conditional gene knock-outs and dynamic events to change gene expression in cells [1]. Therefore, we sought to create a toolkit of these recombinase parts, a fundamental unit of which is the associated target sites for each recombinase. Here, we demonstrate that Rox can recombine with itself when Dre recombinase <partinfo>BBa_K2406081</partinfo> is present.  We then tested its cross-reactivity potential using our measurement constructs, described in the adjacent figure. Essentially, a terminator was flanked by two recombinase target sites. When the recombinase could recognise both sites, it recombination would occur and the terminator would be excised, producing RFP output. This test was useful for two reasons. For one, it demonstrated that our target sites worked as expected, as they would excise their associated target sites. Also, it demonstrated which target sites unexpectedly could cross-react. This is important because researchers have claimed this target site is orthogonal to other popular target sites [1], but this has not been extensively tested in all domains of life.
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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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Our results are summarised on the adjacent figure. We tested Rox self-recognition using <partinfo>Bba_K2406051</partinfo>. We observed recombination with self. This is based on high observed RFP fluorescence output. No cross reactivity with other recombinase target sites was observed, as demonstrated on the graph.
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[[File:Rox Corrected Assays.png |200px|thumb|left|All measurement assays performed involving Rox target sites and corresponding RFP output]]
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==Discussion==
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Our results demonstrate that recombination can efficiently occur between two Rox sites. We observed little cross reactivity with target sites Vox, Slox and Vlox (<partinfo>BBa_K2406001</partinfo>, <partinfo>BBa_K2406003</partinfo>, <partinfo>BBa_K2406002</partinfo>). Therefore, all of these target sites and associated recombinases could be used to catalyse distinct, orthogonal recombination events dynamically within a single cell.
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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 is a target site for Dre tyrosine recombinase. The sequence provided is the "forward" orientation of the Rox target site. If two Rox target sites are present in a DNA molecule and Dre recombinase is present, recombination between the two sites will occur. Two Rox sites forming a direct repeat, that is facing in the same direction, will cause a recombination event that excises the intervening DNA sequence. Rox sites forming an inverted repeat will catalyse recombination that inverts the intervening DNA sequence.
 
  
 
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Latest revision as of 21:40, 28 October 2017


Rox (Dre target site)

Introduction

Dre Recombinase BBa_K2406081 is a tyrosine recombinase that catalyses recombination between Rox target sites [1]. This can lead to integration, excision, or inversion of the DNA sequence in between these target sites. This is an example of a site-specific recombinase (SSR). SSRs have long been recognised to be excellent biological tools, used in conditional gene knock-outs and dynamic events to change gene expression in cells [1]. Therefore, we sought to create a toolkit of these recombinase parts, a fundamental unit of which is the associated target sites for each recombinase. Here, we demonstrate that Rox can recombine with itself when Dre recombinase BBa_K2406081 is present. We then tested its cross-reactivity potential using our measurement constructs, described in the adjacent figure. Essentially, a terminator was flanked by two recombinase target sites. When the recombinase could recognise both sites, it recombination would occur and the terminator would be excised, producing RFP output. This test was useful for two reasons. For one, it demonstrated that our target sites worked as expected, as they would excise their associated target sites. Also, it demonstrated which target sites unexpectedly could cross-react. This is important because researchers have claimed this target site is orthogonal to other popular target sites [1], but this has not been extensively tested in all domains of life.

Schematic outlining principle of all measurement constructs used by Edinburgh_UG 2017

Results

Our results are summarised on the adjacent figure. We tested Rox self-recognition using BBa_K2406051. We observed recombination with self. This is based on high observed RFP fluorescence output. No cross reactivity with other recombinase target sites was observed, as demonstrated on the graph.

All measurement assays performed involving Rox target sites and corresponding RFP output

Discussion

Our results demonstrate that recombination can efficiently occur between two Rox sites. We observed little cross reactivity with target sites Vox, Slox and Vlox (BBa_K2406001, BBa_K2406003, BBa_K2406002). Therefore, all of these target sites and associated recombinases could be used to catalyse distinct, orthogonal recombination events dynamically within a single cell.

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
    COMPATIBLE WITH RFC[25]
  • 1000
    COMPATIBLE WITH RFC[1000]