Difference between revisions of "Part:BBa K215001"

Revision as of 03:42, 1 October 2009

Secretion Signal and Streptavidin Binding Tags

This part acts as the secretion tag to be used with the secretion system BB#, introduced to the registry by the 2009 University of Washington iGEM team. Any favorite protein (afp) can be inserted into the NheI (compatable with XbaI and SpeI, but make sure the resulting product stays in frame) which places the afp in between a series of tags which can be used with the University of Washington 2009 Ideal Protein Purification system (IPP). The series of tags are a Nano tag (binds to streptavidin), a 6x His tag (for IMAC purification), a TEV cute site (for removal of N-terminal tags), the NheI site (insertion of afp), TEV, 6x His tag, the 181 C-terminal amino acids of prtB (secretion signal, secreted by BB#). To use in E. coli we recommend using the composite part BB#, which already has a strong promoter and RBS attached.

Sequence and Features

Assembly Compatibility:

10
COMPATIBLE WITH RFC[10]
12
INCOMPATIBLE WITH RFC[12]
Illegal NheI site found at 115
21
COMPATIBLE WITH RFC[21]
23
COMPATIBLE WITH RFC[23]
25
COMPATIBLE WITH RFC[25]
1000
COMPATIBLE WITH RFC[1000]

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 __NOTOC__
 <partinfo>BBa_K215001 short</partinfo>
-This part is the vector into which afp is inserted to tag it for purification using the IPP system. It contains an NheI restriction site that allows for the insertion of the desired protein coding sequence (NheI is compatible with XbaI and SpeI). This Target Vector part contains two tags: a secretion tag that causes the protein to be exported to the extracellular space, and a nanotag that causes the protein to bind to stretavidin (or some analog).  For this part to function properly, it must be combined with a promoter and RBS, and used in E.coli strain UW5alpha, which has two other plasmids containing the other genes required for proper secretion and display of the protein to be purified on the outer membrane.
+This part acts as the secretion tag to be used with the secretion system BB#, introduced to the registry by the 2009 University of Washington iGEM team.  Any favorite protein (afp) can be inserted into the NheI (compatable with XbaI and SpeI, but make sure the resulting product stays in frame) which places the afp in between a series of tags which can be used with the University of Washington 2009 Ideal Protein Purification system (IPP).  The series of tags are a Nano tag (binds to streptavidin), a 6x His tag (for IMAC purification), a TEV cute site (for removal of N-terminal tags), the NheI site (insertion of afp), TEV, 6x His tag, the 181 C-terminal amino acids of prtB (secretion signal, secreted by BB#).  To use in E. coli we recommend using the composite part BB#, which already has a strong promoter and RBS attached.
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