This composite part was created by cloning the parts BBa_K510000, BBa_K1973033 and BBa_K1973026 together.
nahR (Pseudomonas putida, see BBa_K1031610) is expressed under the constitutive Pr promoter. It encodes a regulatory protein that activates the Psal promoter (see BBa_J61051) in the presence of salicylate. glpF (BBa_K1973026) is expressed from the Psal promoter in the presence of this molecule. The miniTn7 tool allows the stable integration of this module in the chromosome of a wide range of bacteria (see http://parts.igem.org/Genome_Integration).
It was obtained by PCR from a plasmid containing this sequence. The PCR primers added the prefix and suffix needed.
nahR (Pseudomonas putida, see BBa_K1031610) is expressed under the constitutive Pr promoter. It encodes a regulatory protein that activates the Psal promoter (see BBa_J61051) in the presence of salicylate.
We had included the RBS BBa_B0034 in the PCR primers, as well as the scar it should have appeared in the cloning of the DNA piece.
Obtained by PCR from the genome of Pseudomonas aeruginosa PAO1
Codifies the GlpF protein from Pseudomonas aeruginosa, which is a glycerol channel that increases the glycerol intake for bioremediation purposes.
It was amplified by mutagenic PCR. Nucleotide 693 (starting at ATG) was changed (from a cytosine to a thymine). We have inlcuded the sequence corresponding to RBS BBa_B0034 with the PCR primers used for its amplification, as well as the scar it should have been produced by the clonning of both parts.
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.
Conversion of the iGEM parts registry to SBOL2.1