Difference between revisions of "Part:BBa K2406077"
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<partinfo>BBa_K2406077 short</partinfo> | <partinfo>BBa_K2406077 short</partinfo> | ||
| − | + | ==Introduction== | |
| − | This | + | This measurement construct was used to test the cross-reactivity of Vlox and Rox (<partinfo>BBa_K2406002</partinfo>, <partinfo>BBa_K2406000</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 VCre/Vlox and Dre/Rox in one cell. |
| + | [[File:Edinburgh UG measurement constructs.png |200px|thumb|left| 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 Dre <partinfo>BBa_K2406081</partinfo> and VCre<partinfo>BBa_K2406083</partinfo>. We observed no cross-reactivity within this construct, as fluorescence output was negligible when Dre waspresent and not present. | ||
| + | [[File:VCre Assays.png |200px|thumb|left|All measurements performed involving VCre]] | ||
| + | [[File:Dre measurements.png|200px|thumb|left|All measurements performed involving Dre]] | ||
| + | ==Discussion== | ||
| + | The target sites involved in this construct were previously discovered as being potentially orthogonal to other recombinases [1][2]. 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_K2406000</partinfo>. This is because negligible RFP output was seen compared to control. Therefore, the VCre/Vlox and Dre/Rox can be used in an orthogonal manner 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. | ||
| + | [2]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]] | ||
<!-- Add more about the biology of this part here | <!-- Add more about the biology of this part here | ||
Latest revision as of 11:48, 29 October 2017
Vlox-Term-Rox Measurement Construct
Introduction
This measurement construct was used to test the cross-reactivity of Vlox and Rox (BBa_K2406002, BBa_K2406000). 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 VCre/Vlox and Dre/Rox in one cell.
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 Dre BBa_K2406081 and VCreBBa_K2406083. We observed no cross-reactivity within this construct, as fluorescence output was negligible when Dre waspresent and not present.
Discussion
The target sites involved in this construct were previously discovered as being potentially orthogonal to other recombinases [1][2]. 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_K2406000. This is because negligible RFP output was seen compared to control. Therefore, the VCre/Vlox and Dre/Rox can be used in an orthogonal manner 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. [2]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
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal AgeI site found at 886
Illegal AgeI site found at 998 - 1000COMPATIBLE WITH RFC[1000]