Difference between revisions of "Part:BBa K1352006"
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Attaching a FLAG-tag to the end of INP means that it will be expressed on the surface of the cell, the purpose of this is to allow the rapid insertion (via the MCS) of polypeptides (an antigen for example) to the C-terminus end of INP; this allows the surface expression of said polypeptide. | Attaching a FLAG-tag to the end of INP means that it will be expressed on the surface of the cell, the purpose of this is to allow the rapid insertion (via the MCS) of polypeptides (an antigen for example) to the C-terminus end of INP; this allows the surface expression of said polypeptide. | ||
For the 2014 Aberdeen iGEM project, this part was intended to be used as a trypanosome antigen displayer. | For the 2014 Aberdeen iGEM project, this part was intended to be used as a trypanosome antigen displayer. | ||
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+ | Creation of INP-YFP-FLAG fragments followed by InFusion cloning | ||
+ | Four infusion primers (primers with one homologous half (lower case) to the template, and one “overhang” half (upper case), the last 15 nucleotides of which is homologous to another DNA fragment) were designed. | ||
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+ | “INP-VEC-F” (CGCGGCCGCTTCTAGAttaatacgactcactataggg) | ||
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+ | “INP-Rem-R” (aagctttttatcatcatcatctttataatcagatctTCCCGCCACGCTGC) | ||
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+ | “INP-FLAG-F” (AGATCTGATTATAAAGATGATGATGATAAAAAGCTTtaataatactagcaacatatcataacggagtg) | ||
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+ | “INP-VEC-R” (AGCGGCCGCTACTAGTtataaacgcagaaaggccc) | ||
+ | |||
+ | Two INP-YFP-FLAG fragments were created by PCR amplification, both use K523013 as the template, the first uses “INP-VEC-F” and “INP-FLAG-R” primers, the second uses “INP-VEC-R” and “INP-FLAG-F” primers. | ||
+ | The Clontech InFusion kit was used to recombine the two INP-YFP-FLAG fragments with pSB1C3 backbone (cut with Xba1 and Spe1); this kit was followed according to the manufacturer’s instructions. | ||
Revision as of 19:07, 13 October 2014
Ice Nucleation Protein (INP) FLAG-tag
K1352006 is a part which has a FLAG-tag octapeptide flanked by a multiple cloning site (MCS) inserted into Bba_K523013 (K523013 expresses ice nucleation protein (INP)); specifically, on the end of a linker, which is in turn on the C-terminus of INP and is in the same reading frame. The FLAG-tag is a BglII restriction site, followed by a FLAG octapeptide, followed by a HindIII restriction site; the sequence of which is as follows; the octapeptide is uppercase, the restriction sites are lowercase:
(agatctGATTATAAAGATGATGATGATAAAaagctt)
Attaching a FLAG-tag to the end of INP means that it will be expressed on the surface of the cell, the purpose of this is to allow the rapid insertion (via the MCS) of polypeptides (an antigen for example) to the C-terminus end of INP; this allows the surface expression of said polypeptide. For the 2014 Aberdeen iGEM project, this part was intended to be used as a trypanosome antigen displayer.
Creation of INP-YFP-FLAG fragments followed by InFusion cloning
Four infusion primers (primers with one homologous half (lower case) to the template, and one “overhang” half (upper case), the last 15 nucleotides of which is homologous to another DNA fragment) were designed.
“INP-VEC-F” (CGCGGCCGCTTCTAGAttaatacgactcactataggg)
“INP-Rem-R” (aagctttttatcatcatcatctttataatcagatctTCCCGCCACGCTGC)
“INP-FLAG-F” (AGATCTGATTATAAAGATGATGATGATAAAAAGCTTtaataatactagcaacatatcataacggagtg)
“INP-VEC-R” (AGCGGCCGCTACTAGTtataaacgcagaaaggccc)
Two INP-YFP-FLAG fragments were created by PCR amplification, both use K523013 as the template, the first uses “INP-VEC-F” and “INP-FLAG-R” primers, the second uses “INP-VEC-R” and “INP-FLAG-F” primers. The Clontech InFusion kit was used to recombine the two INP-YFP-FLAG fragments with pSB1C3 backbone (cut with Xba1 and Spe1); this kit was followed according to the manufacturer’s instructions.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21INCOMPATIBLE WITH RFC[21]Illegal BglII site found at 1601
- 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal NgoMIV site found at 1036
- 1000COMPATIBLE WITH RFC[1000]