Difference between revisions of "Part:BBa K2406067"

Latest revision as of 12:17, 29 October 2017

Vox-Term-Vlox Measurement Construct

Introduction

This measurement construct was used to test the cross-reactivity of Vox and Vlox (BBa_K2406001, 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 Vika/Vox 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 Vika and VCre recombinases (BBa_K2406082, BBa_K2406083). We observed unexpected cross-reactivity within this construct, as shown by relatively high fluorescence output when Vox and VCre recombinases (BBa_K2406084, BBa_K2406083) were present.

All assays performed involving Vika

All assays performed involving VCre

Discussion

The target sites involved in this construct were not initially tested together and were potentially ortoghonal [1][2]. However, our cross-reactivity tests in E. coli demonstrate they are not orthogonal, unlike the majority of recombinase target sites we tested. Unexpectedly, we observed cross-reactivity between these two target sites BBa_K2406001, BBa_K2406002. Therefore, this indicates that Vika/Vox and VCre/Vlox cannot be used to catalyse distinct and independent recombination events in one 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. [2]Karimova, M., Abi-Ghanem, J., Berger, N., Surendranath, V., Pisabarro, M.T., Buchholz, F. 2013 “Vika/vox, a novel efficient and specific Cre/loxP-like site-specific recombination system”. Nucleic Acids Research 41(2):e37.

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]

@@ Line 2: / Line 2: @@
 __NOTOC__
 <partinfo>BBa_K2406067 short</partinfo>
+==Introduction==
-This part was designed to test if Vox and Vlox Target sites were orthogonal. If either Vika 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.
+This measurement construct was used to test the cross-reactivity of Vox and Vlox (<partinfo>BBa_K2406001</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 Vika/Vox and VCre/Vlox 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 Vika and VCre recombinases (<partinfo>BBa_K2406082</partinfo>, <partinfo>BBa_K2406083</partinfo>). We observed unexpected cross-reactivity within this construct, as shown by relatively high fluorescence output when Vox and VCre recombinases (<partinfo>BBa_K2406084</partinfo>, <partinfo>BBa_K2406083</partinfo>) were present.
+[[File:Vika assays.png |200px|thumb|left|All assays performed involving Vika]]
+[[File:VCre Assays.png|200px|thumb|left|All assays performed involving VCre]]
+==Discussion==
+The target sites involved in this construct were not initially tested together and were potentially ortoghonal [1][2]. However, our cross-reactivity tests in E. coli demonstrate they are not orthogonal, unlike the majority of recombinase target sites we tested. Unexpectedly, we observed cross-reactivity between these two target sites <partinfo>BBa_K2406001</partinfo>, <partinfo>BBa_K2406002</partinfo>. Therefore, this indicates that Vika/Vox and VCre/Vlox cannot be used to catalyse distinct and independent recombination events in one 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.
+[2]Karimova, M., Abi-Ghanem, J., Berger, N., Surendranath, V., Pisabarro, M.T., Buchholz, F. 2013 “Vika/vox, a novel efficient and specific Cre/loxP-like site-specific recombination system”. Nucleic Acids Research 41(2):e37.
+==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