GFP - LasR anchor-inducer protein complex
This part contains a green fluorescent protein (GFP) as a non-reactive chemical compound (anchor) in order to neutralize the active reactive fixed to it, an inducer regulatory protein (LasR is responsible to induce pLas promoter in the presence of 6HSLO3 which is a chemical compound sythesized by an enzyme that LasI gene codes.) and a TEV protease cleavage site between them to assemble these two proteins. BBa_772001 and BBa_K772002 are assembled to be used as the trigger of detection in the whole system which ends with the intracellular transfection (or activation) of TEV protease. After the activation, it is planned that TEV protease will cleave this anchor-inducer protein complex to release free the reporter. In conclusion, in this system, the inducer would activate a transcription pathway on the targeted inducible gene or the reporter would indicate that there is a change in the whole system.
Cleavage site, LasR and GFP sequences are gathered from Registry.
Note that there is an RBS sequence just before the protein sequence in order to eliminate the need of addition of it.
This gene sequence includes an EpCAM (Epithelial Cell Adhesion Molecule) antigen binding domain, OmpA transmembrane protein (dock) and TEV protease C terminus. It is planned to use this subpart with BBa_772, which contains only TEV protease N terminus differently instead of C terminus, in order to induce a signal reaction in the case of EpCAM presence. EpCAM is an natural antigen which is found high significantly in the cases of some breast and colorectal cancer subtypes. On the upstream region of protein sequence, there are a constitutive pLac promoter and appropiate RBS. To see how this part works: Experience.
There are two protein sequences between domains and the dock: One between the binding domain (antibody) and dock and one between the part of TEV protease.
CD225 (EpCAM antibody) is commonly used for the test of presence of EpCAM in the laboratories. The sequences of OmpA and TEV protease terminuses are acquired from the Registry.
TEV tobacco etch virus protease cleavage site
This codes for the seven amino acid sequences that is recognized by the Tobacco Etch Virus protease. The amino acid sequenced used is: Glu-Asn-Leu-Tyr-Phe-Gln-Gly. The TEV protease cuts between Gln and Gly. It is often used in Ni-Nta protein column purifications to cut off the binding tag. This part uses the modified Silver BioBrick prefix and suffix to allow for protein construction.
The TEV cut site is almost always used in conjunction with other peptides, either to release the protein of interest from a binding assay, or to cut two proteins apart post-translationally. Because of this, we used the modified Silver BioBrick prefix and suffix to make the construction of funcitonal fusion peptides possible.
This is the cut sequence for TEV protease put forth by the Dougherty Lab (North Carolina State University). The general form is E-X-X-Y-X-Q-(G/S), but ENLYFQS was found to be most efficient
This gene sequence includes both of EpCAM Binding Docks; BBa_K772001 and BBa_K772002. Thus, this composite part is the main component of CTC (Circulating Tumor Cell) detection system. Docks include an EpCAM (Epithelial Cell Adhesion Molecule) antigen binding domain, OmpA transmembrane protein (dock) and TEV protease C or N terminus. Hopefully, after binding to the EpCAM antigen, TEV protease domains will fix together to form the full structure in order to activate the signalling pathway. (For more information: BBa_K772003) EpCAM is an natural antigen which is found high significantly in the cases of some breast and colorectal cancer subtypes. On the upstream region of protein sequences, there are a constitutive pLac promoter and appropriate RBS. To see how this part works: Experience.
There are two protein sequences between domains and the dock: One between the binding domain (antibody) and dock and one between the domain of TEV protease.
Protein coding region for the firefly [Photinus pyralis] luciferase, codon optimized for yeast.
GFP - LacZ protein complex with flag tags
This part contains a green fluorescent protein (GFP) as a non-reactive chemical compound (anchor) in order to neutralize the active reactive fixed to it, a reporter device (LacZ enzyme catalyses the reaction of the cleavage of X-Gal which results with blue color change) and a TEV protease cleavage site between them to assemble these two proteins. BBa_772001 and BBa_K772002 are assembled to be used as the trigger of detection in the whole system which ends with the intracellular transfection (or activation) of TEV protease. After the activation, it is planned that TEV protease will cleave this anchor-inducer protein complex to release free the reporter. In conclusion, in this system, the inducer would activate a transcription pathway on the targeted inducible gene or the reporter would indicate that there is a change in the whole system. This part contains flag tags extra from BBa_K772006 in the GFP region; because it is planned to be used in the Co-IP/SDS-PAGE assay to be isolated.
Cleavage site, LacZ and GFP sequences are gathered from Registry.
TEV cleaves the following AA sequence with high specificity: * Glu-Asn-Leu-Tyr-Phe-Gln↓Gly but: * Glu-Asn-Leu-Tyr-Phe-Gln↓Ser is also reported Note, the part suggested by the Voigt lab, also contains 2 additional 1xGly flanks for increased accessibility. See also: * BBa_I712077 (TEV N-term) * BBa_I712078 (TEV C-term) * BBa_I712016 (myri+TEV site, Slovenia team igem2007) * BBa_J64007 (Dan Widmaier, Voigt lab) References =========== Dougherty et al. (1989): Molecular genetic analysis of a plant virus polyprotein cleavage site: a model. PMID: 2669323
* gene synthesis * codon optimized for E. coli
This is proteolytic cleavage site for HIV-1 protease.
Protein coding region for the cleavage site of the HCV NS3-4A protease (Part BBa_K1680011).
GFP - LacZ protein generator is generated with the aim of inducing a reporting reaction aftermath of the activation of EpCAM detection system. (BBa_K772100) For more information: BBa_K772006
To achieve a constitutive production, pTetR promoter is added in the upstream region of the original part.
myristoilation signal sequence fused to Tobacco Etch Virus (TEV) protease cleavage site
Signal sequence for myristoilation with proteolytic cleavage site for TEV protease.
GFP - LacZ anchor-inducer protein complex
This part contains a green fluorescent protein (GFP) as a non-reactive chemical compound (anchor) in order to neutralize the active reactive fixed to it, a reporter/inducer device or protein (enzyme etc.) and a TEV protease cleavage site between them to assemble these two proteins. BBa_772001 and BBa_K772002 are assembled to be used as the trigger of detection in the whole system which ends with the intracellular transfection (or activation) of TEV protease. After the activation, it is planned that TEV protease will cleave this anchor-inducer protein complex to release free the inducer/reporter. In conclusion, the inducer would activate a transcription pathway on the targeted inducible gene or the reporter would indicate that there is a change in the whole system.
This sequence encodes a short polypeptide which can be specifically recognized and spliced by DL-Dithiothreitol（DTT）. In our case we add this short sequence between the recombinant protein which we want to purify and the linker protein Spytag, in order to separate the recombinant protein form the Magnetosome.
Our project is to use Magnetosome to purify recombinant protein, and we need to remove the protein from the compound after the protein we are interested combined with Magnetosome. As this short polypeptide can be spliced by DTT, we can meet this need by adding the intein between the recombinant protein and magnetosome.
This sequence comes form the gyrA gene of Mycobacterium xenopi
TEV protease cleaveage site with Standard 25 Prefix/Suffix
This is the cleaveage site for th Tobacco etch virus(TEV) protease commonly used for cleaving fusion proteins. It encodes for the amino acids E N L Y F Q G which are cleaved by the TEV protease. This part also contains the prefix/suffix required for standard 25 assembly into fusion parts.
This part contains the AgeI and NgoMIV sites as part of it's prefix/suffix that allows it to be used in standard 25 assembly. This part was synthesized through PCR oligo synthesis methods using the following primers: Forward 5'---ATTAGAATTCGCGGCCGCTTCTAGATGGCCGGCGAGAATTTGTATTTTCAGGG---3' Reverse 5'---TAATCTGCAGCGGCCGCTACTAGTATTAACCGGTACCCTGAAAATACAAATTCTC---3'
Phan, J., Zdanov, A., Evdokimov, A. G., Tropea, J. E., Peters, H. P. K., Kapust, R. B., Li, M., Wlodawer, A., and Waugh, D. S. (2002). Structural basis for the substrate specificity of tobacco etch virus protease. J. Biol. Chem. 277: 50564-50572.
preScission protease cleavage site (E. coli-optimized)
cleavage site for PreScission??? protease. preScission protease cleavage site (PreScission??? protease is a genetically engineered form of human rhinovirus 3C protease with a GST fusion, allowing for facile cleavage and purification of GST-tagged proteins along with protease removal after recombinant protein cleavage. Enables low-temperature cleavage of fusion proteins containing the eight-residue recognition sequence.) cleavage site: * Leu-Glu-Val-Leu-Phe-Gln↓Gly-Pro References ============== Walker et al. (1994): Efficient and rapid affinity purification of proteins using recombinant fusion proteases. PMID: 7764949
RecA Cleavage Site with Standard 25 Prefix/Suffix
This part encodes for the amino acids V Q A G M F S which are cleavage by the RecA protein within the cell. This sequence was taken from the CI repressor protein which is also cleaved by RecA.
This part contains the AgeI and NgoMIV sites as part of it's prefix/suffix that allows it to be used in standard 25 assembly. This part was synthesized through PCR oligo synthesis methods using the following primers: Forward Primer 5'---ATTAGAATTCGCGGCCGCTTCTAGATGGCCGGCgttCAGGCAGGGATGTTC---3??? Reverse Primer 5???---TAATCTGCAGCGGCCGCTACTAGTATTAACCGGTtgagaacatccctgcctg ---3???
Kim, B, and Little, LB. "LexA and lambda Cl repressors as enzymes: specific cleavage in an intermolecular reaction.." Cell 73.6 (1993): 1165-73. Web. 4 Jul 2011. <http://www.ncbi.nlm.nih.gov/pubmed/8513500>.
GFP - LasR protein complex with flag tags
This part contains a green fluorescent protein (GFP) as a non-reactive chemical compound (anchor) in order to neutralize the active reactive fixed to it, an inducer regulatory protein (LasR is responsible to induce pLas promoter in the presence of 6HSLO3 which is a chemical compound sythesized by an enzyme that LasI gene codes.) and a TEV protease cleavage site between them to assemble these two proteins. BBa_772001 and BBa_K772002 are assembled to be used as the trigger of detection in the whole system which ends with the intracellular transfection (or activation) of TEV protease. After the activation, it is planned that TEV protease will cleave this anchor-inducer protein complex to release free the reporter. In conclusion, in this system, the inducer would activate a transcription pathway on the targeted inducible gene or the reporter would indicate that there is a change in the whole system. This part contains flag tags extra from BBa_K772006 in the GFP region; because it is planned to be used in the Co-IP/SDS-PAGE assay to be isolated.
This gene sequence includes an EpCAM (Epithelial p? Cell Adhesion Molecule) antigen binding domain, OmpA transmembrane protein (dock) and TEV protease N terminus. It is planned to use this subpart with BBa_772, which contains only TEV protease C terminus differently instead of N terminus, in order to induce a signal reaction in the case of EpCAM presence. EpCAM is an natural antigen which is found high significantly in the cases of some breast and colorectal cancer subtypes. On the upstream region of protein sequence, there are a constitutive pLac promoter and appropiate RBS. To see how this part works: Experience.