K215002 drives the expression of [https://parts.igem.org/wiki/index.php?title=Part:BBa_K215001 BBa_K215001] by placing it under the control of a strong, IPTG-inducible promoter (R0011) and RBS (R0034). Any favorite protein (afp) can be inserted into a composite tag [https://parts.igem.org/wiki/index.php?title=Part:BBa_K215001 BBa_K215001]. The composite tag has an NheI site (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 [http://2009.igem.org/Team:Washington University of Washington 2009 Ideal Protein Purification system (IPP)]. The series of tags are, respectively: 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, and the 181 C-terminal amino acids of prtB (secretion tag recognized by the secretion system: [https://parts.igem.org/wiki/index.php?title=Part:BBa_K215107 BBa_K215107]). In order for the secretion tag to signal the secretion of afp the BioBrick [https://parts.igem.org/wiki/index.php?title=Part:BBa_K215107 BBa_K215107] must be present as well.
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This part is GFP (E0040) amplified out and inserted into K215002 at the NheI cut site. For information about this parts functionality when expressed please see [https://parts.igem.org/wiki/index.php?title=Part:BBa_K215002 BBa_K215002] page or the [http://2009.igem.org/Team:Washington University of Washington 2009 project Wiki].
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Cutting a BioBrick coding sequence at X and S and pasting into the NheI site of this construct would result in a stop codon at the resulting scar site. Therefore, to insert a gene into the K215001 of this construct, a PCR product must be generated that keeps the gene of interest in frame with the rest of the sequence. To do this:
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Latest revision as of 00:35, 19 October 2009
Secretion Signal and Streptavidin Binding Tags on GFP
This part is GFP (E0040) amplified out and inserted into K215002 at the NheI cut site. For information about this parts functionality when expressed please see BBa_K215002 page or the [http://2009.igem.org/Team:Washington University of Washington 2009 project Wiki].
