Fast-Fusion GFP-LacZa Reporter (RecA cleavage with Medium 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 RecA protein, which is activated by the presence of damaged DNA, 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 RecA cleavage site (BBa_K648009) followed by a 6 amino acid flexible fusion protein linker (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
Medium 6AA Fusion Protein Linker: GGSGGS with Standard 25 Prefix/Suffix
The inspiration for this part came from the linker used in the 2010 Imperial College London iGEM team GFP-Xyle fusion part: BBa_K316007 Other Sources: 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 mid-sized linker of the three fusion protein linkers created by the Penn State 2011 iGEM team. It encodes for the six amino acids GGSGGS 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: 5??? TATTGAATTCGCGGCCGCTTCTAGATGGCCGGCggaggttcagg 3??? Reverse: 5???AATACTGCAGCGGCCGCTACTAGTATTAACCGGTgctgcctcctgaacctccGC 3'
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???
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