Fast-Fusion GFP-LacZa Reporter (TEV cleavage with long linker)
The design of this part was inspired by the Imperial College London 2010 iGEM Team's Fast-Response module, part BBa_K316007.
This is a fast-acting reporter using the LacZa gene (BBa_K648012) fused to GFP (BBa_K648013). In its normal un-cleaved state the polymerization ability necessary for enzymatic action is inactivated. Once the fusion protein is cleaved by the TEV protease the B-gal enzyme encoded by the LacZa gene is able to convert X-gal to galactose and 5-bromo-4-chloro-3-hydroxyindole, the latter of which is oxidized to the dark blue product 5,5'-dibromo-4,4'-dichloro-indigo. This reporter is able to produce a visible blue color response in a matter of minutes. This version uses the TEV cleavage site (BBa_K648008) followed by the smallest of the three flexible fusion protein linkers (BBa_K648005). The linker is attached to the N-terminus of the LacZa gene.
All parts were assembled via standard 25 assembly methods
RBS (Elowitz 1999) -- defines RBS efficiency
Varies from -6 to +1 region from original sequence to accomodate BioBricks suffix. No secondary structures are formed in the given RBS region. Users should check for secondary structures induced in the RBS by upstream and downstream elements in the +50 to -50 region, as such structures will greatly affect the strength of the RBS. Contact info for this part: Brian Chow
Vinay S Mahajan, Voichita D. Marinescu, Brian Chow, Alexander D Wissner-Gross and Peter Carr IAP, 2003.
Double terminator consisting of BBa_B0010 and BBa_B0012
Long 10AA Fusion Protein Linker with Standard 25 Prefix/Suffix
Patrick Argos, An investigation of oligopeptides linking domains in protein tertiary structures and possible candidates for general gene fusion, Journal of Molecular Biology, Volume 211, Issue 4, 20 February 1990, Pages 943-958, ISSN 0022-2836, DOI: 10.1016/0022-2836(90)90085-Z. (http://www.sciencedirect.com/science/article/pii/002228369090085Z) Ryoichi Arai,Hiroshi Ueda,Atsushi Kitayama,Noriho Kamiya,and Teruyuki Nagamune. Design of the linkers which effectively separate domains of a bifunctional fusion protein Protein Eng. (2001) 14(8): 529-532 doi:10.1093/protein/14.8.529
This is the longest of the three fusion protein linkers created by the Penn State 2011 iGEM team. It encodes for the ten amino acids GENLYFQSGG and has a prefix and suffix compatible with assembly standard 25. It is used in one of the variants of our Fast-Fusion protein reporter system.
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: TATTGAATTCGCGGCCGCTTCTAGATGGCCGGCggtgaaaatttgtattttcaa Reverse: AATACTGCAGCGGCCGCTACTAGTATTAACCGGTaccaccagattgaaaatacaaattttcacc
Jim Rose, Alex Bina, Ben Alouidor, Brian Avison
TEV protease cleaveage site with Standard 25 Prefix/Suffix
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.
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'
This part was amplified out of registry stock of LacZa in order to include the standard 25 prefix/suffix
This is the lacZa subunit used to complement cells for blue/white screening. It has been cloned into a vector including the prefix/suffix required for standard 25 assembly for fusion proteins.
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. It was synthesized through PCR oligo synthesis methods using the following primers: Forward TATTGAATTCGCGGCCGCTTCTAGATGGCCGGCACCATGATTACGGATTCAC (57.74 oC) Reverse ATAACTGCAGCGGCCGCTACTAGTATTAACCGGTTCACTCCAGCCAGC (55.05 oC)
This part is similar to the part BBa_E0040 commonly used as a reporter.
This is the standard GFP protein reporter (E0040) cloned with the prefix/suffix required for standard 25 assembly. It can easily be used to create fusion proteins through this method.
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. It was synthesized through PCR oligo synthesis methods using the following primers: Forward (56.88 degrees) TATTGAATTCGCGGCCGCTTCTAGATGGCCGGCCGTAAAGGAGAAGAACTTTTC Reverse (56.95 degrees) AATACTGCAGCGGCCGCTACTAGTATTAACCGGTCTTGTCGTCATCATCTTTATAAT
Derived from the TE terminator of T7 bacteriophage between Genes 1.3 and 1.4 <genbank>V01146</genbank>.
Transcription terminator for the E.coli RNA polymerase.
Suggested by Sri Kosuri and Drew Endy as a high efficiency terminator. The 5' end cutoff was placed immediately after the TAA stop codon and the 3' end cutoff was placed just prior to the RBS of Gene 1.4 (before AAGGAG). Use anywhere transcription should be stopped when the gene of interest is upstream of this terminator.
Transcriptional terminator consisting of a 64 bp stem-loop.
Conversion of the iGEM parts registry to SBOL2.1