Difference between revisions of "Part:BBa K2586004"

Revision as of 08:08, 13 September 2018

Strep-Tag-aroE: aroE linked to a Strep-Tag

This part is the combination of (BBa_K2586003) with a Strep-Tag.
The Strep-protein can be used in (SPINE): Strep-protein interaction experiments. SPINE is a powerful tool to detect protein-protein interactions in vivo . The bait protein (aroE in this case) has to be expressed with a Strep-tag under the conditions when the potential interaction partners are presumably present in the cells. The addition of formaldehyde (FA), which easily permeates the cell wall and membrane, links the bait protein to its potential interaction partners.
The bait protein together with its potential interaction partners can then be isolated using a Strep-Tactin sepharose column by eluting with desthiobiotin- or biotin-containing buffer.
The cross-links between the bait protein and the potential interaction partner can be cleaved by heating the samples in Laemmli buffer. Finally, the co-purified interaction partner(s) can be separated by [http://www.ruf.rice.edu/~bioslabs/studies/sds-page/gellab2.html (SDS PAGE)], identified in mass spectrometry and Western Blotting.

Sequence and Features

Assembly Compatibility:

10
INCOMPATIBLE WITH RFC[10]
Illegal prefix found in sequence at 1
Illegal suffix found in sequence at 1390
Illegal EcoRI site found at 74
Illegal PstI site found at 1232
12
INCOMPATIBLE WITH RFC[12]
Illegal EcoRI site found at 1
Illegal EcoRI site found at 74
Illegal NheI site found at 24
Illegal NheI site found at 192
Illegal SpeI site found at 1391
Illegal PstI site found at 1232
Illegal PstI site found at 1405
Illegal NotI site found at 7
Illegal NotI site found at 1398
21
INCOMPATIBLE WITH RFC[21]
Illegal EcoRI site found at 1
Illegal EcoRI site found at 74
Illegal BamHI site found at 95
23
INCOMPATIBLE WITH RFC[23]
Illegal prefix found in sequence at 1
Illegal suffix found in sequence at 1391
Illegal EcoRI site found at 74
Illegal PstI site found at 1232
25
INCOMPATIBLE WITH RFC[25]
Illegal prefix found in sequence at 1
Illegal EcoRI site found at 74
Illegal XbaI site found at 16
Illegal SpeI site found at 1391
Illegal PstI site found at 1232
Illegal PstI site found at 1405
Illegal NgoMIV site found at 517
1000
INCOMPATIBLE WITH RFC[1000]
Illegal BsaI.rc site found at 60

@@ Line 4: / Line 4: @@
 This part is the combination of [https://parts.igem.org/Part:BBa_K2586003 (BBa_K2586003)] with a <i>Strep</i>-Tag. <br/>
-The Strep-protein can be used in [https://en.wikipedia.org/wiki/SPINE_(molecular_biology (SPINE)]:Strep-protein interaction experiments. SPINE is a powerful tool to detect protein-protein interactions <i>in vivo </i>. The bait protein (<i>aroE</i> in this case) has to be expressed with a <i>Strep</i>-tag under the conditions when the potential interaction partners are presumably present in the cells. The addition of formaldehyde (FA), which easily permeates the cell wall and membrane, links the bait protein to its potential interaction partners. <br/>
+The Strep-protein can be used in [https://en.wikipedia.org/wiki/SPINE_(molecular_biology (SPINE)]: Strep-protein interaction experiments. SPINE is a powerful tool to detect protein-protein interactions <i>in vivo </i>. The bait protein (<i>aroE</i> in this case) has to be expressed with a <i>Strep</i>-tag under the conditions when the potential interaction partners are presumably present in the cells. The addition of formaldehyde (FA), which easily permeates the cell wall and membrane, links the bait protein to its potential interaction partners. <br/>
 The bait protein together with its potential interaction partners can then be isolated using a <i>Strep</i>-Tactin sepharose column by eluting with desthiobiotin- or biotin-containing buffer. <br/>
 The cross-links between the bait protein and the potential interaction partner can be cleaved by heating the samples in Laemmli buffer. Finally, the co-purified interaction partner(s) can be separated by [http://www.ruf.rice.edu/~bioslabs/studies/sds-page/gellab2.html (SDS PAGE)], identified in mass spectrometry and Western Blotting.