Difference between revisions of "Part:BBa K215002"
| Line 4: | Line 4: | ||
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 (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: [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. | 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 (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: [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. | ||
| − | Cutting a BioBrick coding sequence at X and S and pasting into the NheI site of this construct | + | 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: |
#Design a primer that complements the coding sequence of interest starting at the second amino acid and ending in frame before the stop codon. | #Design a primer that complements the coding sequence of interest starting at the second amino acid and ending in frame before the stop codon. | ||
| − | ##Forward Primer: <8 random bp's> - <NheI | + | ##Forward Primer: <8 random bp's> - <NheI or XbaI or SpeI> - <15-21bp complementing your gene of interest, starting at the second codon> |
| − | ##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 | + | ##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 or XbaI or SpeI> - <8 random bp's> |
#Amplify the gene of interest | #Amplify the gene of interest | ||
Revision as of 18:36, 1 October 2009
pLac+RBS+Secretion Signal and Streptavidin Binding Tags
K215002 drives the expression of 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 BBa_K215001. The composite tag has an 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: BBa_K215107). In order for the secretion tag to signal the secretion of afp the BioBrick BBa_K215107 must be present as well.
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:
- Design a primer that complements the coding sequence of interest starting at the second amino acid and ending in frame before the stop codon.
- Forward Primer: <8 random bp's> - <NheI or XbaI or SpeI> - <15-21bp complementing your gene of interest, starting at the second codon>
- 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 or XbaI or 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
- 10COMPATIBLE WITH RFC[10]
- 12INCOMPATIBLE WITH RFC[12]Illegal NheI site found at 196
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]