pUC18R6KT-miniTn7BB-Gm-improved_flipflop_(module I)
The pUC18R6KT vector was amplified by PCR using as template a pTNS2 plasmid (GenBank accession number: AY884833) provided by Herbert P. Schweizer. The primers were designed to flanked the pUC18R6KT with SfiI restriction sites and allow the insertion of the mini-Tn7-Gm. Primers: attcGGCCTAGGCGGCCgtcgttttacaacgtcgtgac and attcGGCCGCCTAGGCCggaagcataaagtgtaaagcctg. The miniTn7BB-Gm minitransposon was synthesized commercially, and then digested at the flanking SfiI sites and cloned into SfiI-digested pUC18R6KT PCR products. The improved flip-flop (module I) was synthesized commercially too.
This plasmid allows the integration of the improved flip-flop (module I) (part: BBa_K51019) into bacterial chromosomes using the pUC18Sfi-miniTn7BB-Gm (BBa_K51012) characteristics. Single copy may improve the function of regulatory circuits, as bistable systems. For more information about the function of the miniTn7 BioBrick toolkit and flip-flops, visit the iGEM team UPO-Sevilla 2011 wiki.
The BBa_K510019 BioBrick was inserted within the BCS of pUC18R6KT-miniTn7BB-Gm (BBa K510012) by EcoRI and PstI clonning.
The pUC18R6KT vector was amplified by PCR using as template a pTNS2 plasmid (GenBank accession number: AY884833) provided by Herbert P. Schweizer. The primers were designed to flanked the pUC18R6KT with SfiI restriction sites and allow the insertion of the mini-Tn7-Gm. Primers: attcGGCCTAGGCGGCCgtcgttttacaacgtcgtgac and attcGGCCGCCTAGGCCggaagcataaagtgtaaagcctg. The miniTn7BB-Gm minitransposon was synthesized commercially, and then digested at the flanking SfiI sites and cloned into SfiI-digested pUC18R6KT PCR products.
pUC18R6KT-miniTn7BB-Gm is a vehicle vector for the minitransposon miniTn7BB-Gm. This transposon is a part of the miniTn7 BioBrick toolkit, a set of plasmids harboring Tn7 derivatives that can be used for integration of BioBricks in single copy at a conserved target (the attTn7 site), in multiple bacteria. The structure of the mini-Tn7BB-Gm transposon is shown in the figure below. Tn7R and Tn7L are the right and left ends of Tn7 transposon, respectively, required for recognition of the transposase machinery and transposition. FRT is the Flp recombinase target site for excision of the resistance once the transposon is inserted in the genome of a living organism. The gentamycin resistance cassette (Gm) is flanked by restriction sites in order to facilitate the generation of variants with different antibiotic resistance markers. The BioBrick cloning site (BCS) includes the prefix and suffix restrictions sites used in assembly standard 10, and it is the place where BioBricks are inserted for genome integration. This plasmid is replicative only in E. coli strains expressing the pir gene thanks to its R6K replication origin. Also it has an origin for transference (oriT) for conjugation purposes. This plasmid can be used to maintain the miniTn7-Gm transposon in E. coli pir+ strains and may be used for transposition into the genomes of any bacteria without the pir gen, in which it is non-replicative. As a BioBrick cloning vector, pUC18R6KT-miniTn7BB-Gm is fully compatible with Assembly Standard 10. The complete transposon is flanked by SfiI restriction sites to facilitate subcloning in other vectors.
Kyoung-Hee Choi, Jared B Gaynor, Kimberly G White, Carolina Lopez, Catharine M Bosio, RoxAnn R Karkhoff-Schweizer & Herbert P Schweizer (2005). A Tn7-based broad-range bacterial cloning and expression system. Nature Methods, vol.2 NO.6, June 2005, 443.
It's a part which comes from different genes forming the bassic flip-flop with anothers improvements.
Here we show the final structure of our improved bistable. As well as the two operons with their protein fusions and the promoter with the asRNA, we show the different restriction sites we have introduced and the enzymes that target each site. Finally, the total length of our construction is indicated in the figure.
Victor Alvarez, Felix Reyes and Yolanda Gonzalez
Conversion of the iGEM parts registry to SBOL2.1