Team:Freiburg/Parts

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All of our parts feature our expanded Pre- and Suffix for in frame cloning of fusion proteins [http://parts.mit.edu/igem07/index.php/Freiburg07/report_fusion_parts (more)]. A complete, detailed list of all parts submitted to the registry in this year´s project can be found [http://partsregistry.org/cgi/partsdb/pgroup.cgi?pgroup=iGEM2008&group=Freiburg here], as well as the respective DNA-Sequences.

basic parts

• Signal peptide
  

Part Name:
[http://partsregistry.org/wiki/index.php?title=Part:BBa_K157001 BBa_K157001]
Description:
This sequence mediates transportation of the protein to a translocational pore by an RNA–multiprotein complex, the signal recognition particle (SRP) when fused to the n-terminus of a fusion protein with a transmembrane region[1]. There, after a pause of translation, the signal sequence is released and translation and translocation of the nascent chain are restarted[2].
Source:
Human EGFR (ErbB-1) signal sequence; originally mediating membrane integration of the EGF-receptor (ErbB-1). Sequence taken from UniProtKB/Swiss-Prot entry P00533.
Gene synthesis by ATG:biosynthetics, optimized for expression in homo sapiens by iGEM-Team Freiburg 2008.
References:
[1] Walter P, Johnson AE: “Signal sequence recognition and protein targeting to the endoplasmic reticulum membrane.” Annu Rev Cell Biol 1994, 10:87-119
[2] Robert M Stroud, Peter Walter: “Signal sequence recognition and protein targeting”, Current Opinion in Structural Biology 1999, 9:754–759
AA sequence:
MRPSGTAGAALLALLAALCPASRA

• Erbb1-transmembrane region
  

Part Name:
[http://partsregistry.org/wiki/index.php?title=Part:BBa_K157002 BBa_K157002]
Description:
Helical, single-span transmembrane region of the human EGF-Receptor type ErbB-1, sequence taken from UniProtKB/Swiss-Prot entry P00533.
source:
Transmembrane region of the human EGF-Receptor type ErbB-1. Sequence taken from UniProtKB/Swiss-Prot entry P00533. Gene synthesis by ATG:biosynthetics, optimized for expression in homo sapiens by iGEM-Team Freiburg 2008.
AA sequence:
IATGMVGALLLLLVVALGIGLFM

• Sc-Fv anti NIP
  

Part Name:
Bba_K157003
Description:
Singlechain Fv.Fragment of the Anti-Nitro-Iodo-Phenol-antibody B1-8. Binds the hapten NIP (Nitro-Iodo-Phenole) with an affinity of 2.0 uM[1,2]. Designed for fusion to proteins or peptides via in frame cloning.
source:
Gene synthesis by GeneArt, optimized for expression in homo sapiens.
References:
[1]Ana Cumano and Klaus Rajewski: “Clonal recruitment and somatic mutation in the generation of immunological memory to the hapten NP”, The EMBO Journal vol. 5 no.10 pp. 2459-2468, 1986
[2]D. Allen, T. Simon, F. Sablitzky, K. Rajewski and A. Cumano: “Antibody engineering for the analysis of affinity maturation of an anti-hapten response”, The EMBO Journal vol. 7 no.7 pp. 1995-2001, 1988
AASequence:
QVQLQQPGAELVKPGASVKLSCKASGYTFTSYWMHWVKQRPGRGLEWIGRIDPNSGGTKYNEKFKSKATLTVDKPSSTAYMQLSSLTSEDSAV YYCARYDYYGGSYFDYWGQGTTVTVSSGGGGSGGGGSGGGGSQAVVTQESALTTSPGETVTLTCRSSTGAVTTSNYANWVQEKPDHLFTGLIG GTNNRAPGVPARFSGSLIGDKAALTITGAQTEDEAIYFCALWYSNHWVFGGGTKLTVLG

• LipocalinFluA
  

Part Name:
Bba_K157004
Description:
Fluoresceine A -binding derivative of a Lipocalin (BBP, bilin binding protein from pieris brassicae), originally designed by Arne Skerra[1,2].
Source:
Gene synthesis by GeneArt, optimized for expression in homo sapiens.
References:
[1] Gerald Beste, Frank S. Schmidt, Thomas Stibora and A. Skerra: “Small antibody-like proteins with prescribed ligand specificities derived from the lipocalin fold“,Proc. Natl. Acad. Sci. USA Vol. 96, pp. 1898–1903, March 1999 Biochemistry
[2]Ingo P. Korndörfer, Gerald Beste and A. Skerra: “Crystallographic Analysis of an “Anticalin” With Tailored Specificity for Fluorescein Reveals High Structural Plasticity of the Lipocalin Loop Region”, PROTEINS: Structure, Function, and Bioinformatics 53:121–129 (2003)
AA sequence:
MAGDVYHDGA CPEVKPVDNF DWSQYHGKWW EVAKYPSPNG KYGKCGWAEY TPEGKSVKVS RYDVIHGKEY FMEGTAYPVG DSKIGKIYHS RTVGGYTRKT VFNVLSTDNK NYIIGYSCRY DEDKKGHWDH VWVLSRSMVL TGEAKTAVEN YLIGSPVVDS QKLVYSDFSE AACKVNNTG

• Split-Cerulean-C-CFP
  

Part Name:
Bba_K157005
Description:
C-terminal fragment (Amino acids156-236) of the monomeric[5] cyan fluorescent protein variant “Cerulean”, designed for fusion/BiFC[1,3]. Reassembly with Split-Cerulean-nCFP (Part Bba_K157006) generates the complete, working CFP “Cerulean”, whereas combination with the N-terminal fragment (Part Bba_K157008) of the monomeric yellow fluorescent Protein “Venus”[4] (Part Bba_I757008) results in green fluorescence[2].
AASequence:

• Split-Cerulean-N-CFP
  

Part Name:
Bba_K157006
Description:
N-terminal fragment (Amino acids1-155) of the monomeric[5] cyan fluorescent protein variant “Cerulean”, designed for fusion/BiFC[1,3]. Reassembly with Split-Cerulean-cCFP (Part Bba_K157005) generates the complete, working CFP “Cerulean”, whereas combination with the C-terminal fragment (Part Bba_K157007) of the monomeric yellow fluorescent Protein “Venus”[4] (Part Bba_I757008) results in green fluorescence[2].
AASequence:

• Split-Venus-C-YFP
  

Part Name:
Bba_K157007
Description:
C-terminal fragment (Amino acids157-237) of the monomeric[5] yellow fluorescent protein variant “Venus” [4], designed for fusion/BiFC[1,3]. Reassembly with Part Bba_K157008 (Split-Venus-nYFP) generates the complete, working YFP “Venus”, whereas combination with the n-terminal fragment (Part Bba_K157006) of the monomeric cyan fluorescent Protein “CeruleanR206K” (Part Bba_I757009) results in green fluorescence[2].
AASequence:
KNGIKANFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSKLSKDPNEKRDH

• Split-Venus-N-CFP
  

Part Name:
Bba_K157008
Description:
N-terminal fragment (Amino acids1-156) of the monomeric[5] yellow fluorescent protein variant “Venus” [4], designed for fusion/BiFC[1,3]. Reassembly with Part Bba_K157007 (Split-Venus-cYFP) generates the complete, working YFP “Venus”, whereas combination with the C-terminal fragment (Part Bba_K157005) of the monomeric cyan fluorescent Protein “CeruleanR206K” (Part Bba_I757009) results in green fluorescence[2].
AASequence:
MSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKFICTTGKLPVPWPTLVTTLGVQCFARYPDHMKRHDFFKSAMPEG YVQERTIFFKDDGNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNAISDNVYITADKQ

Source (all split-fluorophores):
Gene synthesis by GeneArt, optimized for expression in homo sapiens.
References (all split-fluorophores):
- [1] Chang-Deng Hu, Yurii Chinenov, Tom K. Kerppola: ”Visualization of Interactions among bZIP and Rel Family Proteins in Living Cells Using Bimolecular Fluorescence Complementation”, Molecular Cell, Vol. 9, 789–798, April, 2002
- [2] Chang Deng Hu, Tom K. Kerppola: “Simultaneous visualization of multiple protein interactions in living cells using multicolor fluorescence complementation analysis”, Nat Biotechnol. 2003 May; 21(5):539-545 (doi:10. 1038/nbt816)
-[3] Tom K. Kerppola: “Design and implementation of bimolecular fluorescence complementation (BiFC) assays for the visualization of protein interactions in living cells”, Nat Protoc. 2006;1(3):1278-1286 (doi:10.1038/nprot.2006.201)
-[4]Nagai, T. et al. “A variant of yellow fluorescent protein with fast and efficient maturation for cell-biological applications” J. Biol. Chem. 276, 29188-29194, 2001
-[5]Roger Y. Tsien et al. „Creating new fluorescent probes for cell biology“, Nature Biotechnology Reviews, Vol. 3, 906-918, 2002

• Split-Fluo-Linker
  

Part Name:
Bba_K157009
description:
Originally, this linker was used for fusion to the N-terminus of the C-terminal half of split fluorophores: Protein interactions can be examined by BiFC[1] using complementary, non-fluorescent fragments of fluorophores[2]. Therefore, it is essential that the C-terminal fragment of the fluorophore is not restricted too much in its mobility. The linker allows orientation and adaption of the C-terminal fragment to the N-terminal fragment of the split fluorophore and, thus, the reassembly of a working fluorescent protein. It has already been used in BiFC assays and is known to serve this purpose well[3]; anyway, another, more flexible linker that could be used instead is our “GGGGS-linker” (Part Bba_K157010).
References:
- [1] Chang-Deng Hu, Yurii Chinenov, Tom K. Kerppola: ”Visualization of Interactions among bZIP and Rel Family Proteins in Living Cells Using Bimolecular Fluorescence Complementation”, Molecular Cell, Vol. 9, 789–798, April, 2002
- [2] Chang Deng Hu, Tom K. Kerppola: “Simultaneous visualization of multiple protein interactions in living cells using multicolor fluorescence complementation analysis”, Nat Biotechnol. 2003 May; 21(5):539-545 (doi:10. 1038/nbt816)
-[3] Tom K. Kerppola: “Design and implementation of bimolecular fluorescence complementation (BiFC) assays for the visualization of protein interactions in living cells”, Nat Protoc. 2006;1(3):1278-1286 (doi:10.1038/nprot.2006.201)
source:
Gene synthesis by ATG:biosynthetics, optimized for expression in homo sapiens by iGEM-Team Freiburg 2008.
AA Sequence:
RPACKIPNDLKQKVMNH

• GGGGS-LINKER_BCR-Transmem.
  

Part Name:
Bba_K157010
Description:
Helical single-span transmembrane region of the B-Cell-Receptor with a flexible 15 aa Linker at the N-terminus. Designed for fusion to proteins or peptides that are to be presented at the cells surface; signal peptide for membrane integration (e.g. part Bba_K157001) required at the N-terminus of the whole construct!

• His-Tag (improved)
  

Part Name:
Bba_K157011
See Part BBa_I757013 (Freiburg 2007)

• Strep-Tag II (improved)
  

Part Name:
Bba_K157012
See Part BBa_I757014 (Freiburg 2007)

• GGGS-linker (pMA)
  

Part Name:
Bba_K157013
Description:
A flexible, 15 aa long linker designed for fusion to/of proteins or peptides via in frame cloning. Consists of Glycine and Serine only.
source:
Gene synthesis by ATG:biosynthetics, optimized for expression in homo sapiens by iGEM-Team Freiburg 2008.
AA sequence:
GGGSGGGSGGGSGGG

composite parts

Part BBa_K157014 - BBa_K157039, Part BBa_K157040 (modified expression plasmid with CMV-promoter(for expression in human cells) and BBa_K157041 (BBa_K157040 + CFP)