Difference between revisions of "Part:BBa K215002"

Revision as of 05:36, 1 October 2009

pLac+RBS+Secretion Signal and Streptavidin Binding Tags

Any favorite protein (afp) can be inserted into the NheI (compatible with XbaI and SpeI, but make sure the resulting product stays in frame, described below) 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 tag recognized by the secretion system: BB#). In order for the secretion tag to signal the secretion of afp the BioBrick # must be present as well.

Cutting a BioBrick coding sequence at X and S and pasting into the NheI site of this construct will result in a stop codon at the resulting scar site. Therefore, to insert a gene a PCR product must be generated that keeps the gene of interest in frame with the rest of the sequence. To do this:

Design a primer that complements the coding sequence of interest starting at the second amino acid and ending in frame before the stop codon.
1. Forward Primer: <8 random bp's> - <NheI/XbaI/SpeI> - <15-21bp complementing your gene of interest, starting at the second codon>
2. Reverse Primer: <15-21bp complementing your gene of interest, ending BEFORE the stop codon (make sure it is a muliple of three to stay in frame)> - <NheI/XbaI/SpeI> - <8 random bp's>

Amplify the gene of interest
Clone into the NheI site of this construct.
Screen colonies with either the Forward Primer+VR or the VF2+Reverse primer for inserts in the appropriate direction.
Your fusion protein is tagged and ready for secretion and streptavidin binding!

Sequence and Features

Assembly Compatibility:

10
COMPATIBLE WITH RFC[10]
12
INCOMPATIBLE WITH RFC[12]
Illegal NheI site found at 196
21
COMPATIBLE WITH RFC[21]
23
COMPATIBLE WITH RFC[23]
25
COMPATIBLE WITH RFC[25]
1000
COMPATIBLE WITH RFC[1000]

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 <partinfo>BBa_K215002 short</partinfo>
-Any favorite protein (afp) can be inserted into the NheI (compatible 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 tag recognized by the secretion system: BB#). To use in E. coli we recommend using the composite part BB#, which already has a strong promoter and RBS attached. In order for the secretion tag to function this part needs to be used in conjunction with BB#.
+Any favorite protein (afp) can be inserted into the NheI (compatible with XbaI and SpeI, but make sure the resulting product stays in frame, described below) 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 tag recognized by the secretion system: BB#). In order for the secretion tag to signal the secretion of afp the BioBrick # must be present as well.
 Cutting a BioBrick coding sequence at X and S and pasting into the NheI site of this construct will result in a stop codon at the resulting scar site.  Therefore, to insert a gene a PCR product must be generated that keeps the gene of interest in frame with the rest of the sequence.  To do this: