Fast-Fusion GFP-Xyle Reporter (RecA cleaveage with small 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 Xyle gene (BBa_K648011) fused to GFP. In its normal un-cleaved state the polymerization ability necessary for enzymatic action is inactivated. Once the fusion protein is cleaved by the RecA, which is activated by damaged DNA, the catechol-2,3-dioxygenase enzyme encoded by the Xyle gene is able to convert catechol to the bright yellow product 2-hydroxy-cis,cis-muconic semialdehyde. This reporter is able to produce a visible color response in a matter of minutes. This version uses the RecA cleavage site (BBa_K648009) followed by the smallest of the three flexible fusion protein linkers (BBa_K648005). The linker is attached to the N-terminus of the Xyle 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
Short Fusion Protein Linker: GGSG 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 shortest of the three fusion protein linkers created by the Penn State 2011 iGEM team. It encodes for the four amino acids GGSG 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 (57.78 degrees) TATTGAATTCGCGGCCGCTTCTAGATGGCCGGCggtggttctggtACC Reverse (57.78 degrees) AATACTGCAGCGGCCGCTACTAGTATTAACCGGTaccagaaccaccG
Jim Rose, Alex Bina, Ben Alouidor, Brian Avison
RecA Cleavage Site with Standard 25 Prefix/Suffix
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>.
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???
The original genetic material for this part came from part BBa_K316007 which was mutated via PCR based site-directed mutagensis. The xyle portion of this large part was then amplified via PCR reaction and cloned into a new vector along with a prefix/suffix for standard 25 assembly.
This is a mutated version of the Xyle reporter gene which encodes for the enzyme catechol-2,3-dioxygenase (metapyrocatechase), which converts catechol to the bright yellow product 2-hydroxy-cis,cis-muconic semialdehyde. This version of Xyle has been made compatible with standard 25 assembly methods by removing three restriction sites (two NgoMIV sites: at bp 315 and 486, as well as one AgeI site: at bp 837). These mutations were made synonymous with the original sequence and codon optimized for E. Coli. Because of the synonymous mutations, this gene can be easily used to create fusion protein parts. For more information on the Xyle gene and its uses as a reporter see part BBa_J33204.
The mutations in this gene were made so that they were synonymous and codon optimized for E. coli cells. The part also contains the prefix/suffix for standard 25 assembly with addition restriction sites NgoMIV and AgeI.
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