pUC18Sfi-miniTn7BB-Gm-improved_flipflop_(module I)
The miniTn7BB-Gm minitransposon was synthesized commercially and pUC18SfiI is a commercial vector. The improved flip-flop (module I) was synthesized commercially.
This plasmid allows the integration of the improved flip-flop (module I) (part: BBa_K510019) into bacterial chromosomes using the pUC18Sfi-miniTn7BB-Gm (BBa_K51000) characteristics. Single copy may improve the function of regulatory circuits, as bistable systems. For more information about the function of the miniTn7 BioBrick toolkit, visit the iGEM team UPO-Sevilla 2011 wiki.
The BBa_K510019 BioBrick was inserted within the BCS of pUC18Sfi-miniTn7BB-Gm (BBa K510000) by EcoRI and PstI clonning. In order to construct the pUC18Sfi-miniTn7BB-Gm delivery plasmid, the miniTn7BB-Gm transposon was cleaved from the commercial plasmid pMA (Mr. Gene) with SfiI and ligated to SfiI-digested pUC18Sfi. This strategy removes all multi-cloning restriction sites from pUC18Sfi, except for the flanking SfiI, thus guaranteeing that the unique sites in the transposon are not duplicated. The remove of the multi-cloning restriction sites was verified by analytic digestions.
The pUC18-SfiI cloning vector was kindly provided by the Microbiology laboratory of Centro Andaluz de Biolog??a del Desarrollo (CABD) in Sevilla. The miniTn7BB-Gm synthetic transposon was designed by the iGEM 2011 team UPO-Sevilla based on the set of miniTn7 derivatives developed by Choi et al. (2005) and commercially synthesized by Mr. Gene (Regensburg, Germany). K.-H. Choi, J. B. Gaynor, K. G. White, C. Lopez, C. M. Bosio, R. R. Karkhoff-Schweizer & H. P. Schweizer (2005). A Tn7-based broad-range bacterial cloning and expression system. Nature Methods, vol.2 NO.6, June 2005, 443.
The miniTn7BB-Gm synthetic minitransposon was digested at the flanking SfiI sites and cloned into SfiI-digested pUC18-Sfi. This results in the elimination of the complete pUC18-SfiI multi-cloning site, except for the duplicated SfiI sites that remain on both sides of the transposon, thus facilitating its transfer to other vectors. This plasmid can be used to keep the mini-Tn7-Gm in E. coli and other enterics, and may be used for transposition into the genomes of non-enteric bacteria, in which it is non-replicative. 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. The complete construction is flanked by SfiI restriction sites to facilitate cloning.
In order to make this vector compatible with assembly standard 10, several restriction sites were removed from the synthetic sequence.
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