Basic parts were cloned into the miniTn7BB-Gm Backbone
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. nasF (see BBa_K1973001) is an attenuator of transcription elongation (terminator) from Klebsiella pneumoniae that decreases the basal expression from the Psal promoter. xylS2 (BBa_K1973002) codifies the modified XylS2 protein from Pseudomonas putida MT1, which is a transcriptional activator of the Pm promoter in response to salicylate. This module provides a tight expression regulation by salicylate thanks to a two-step (NahR and XylS2) system. 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).
Designs are described in basic parts pages
It was obtained by PCR from a plasmid containing its sequence.
Codifies the modified XylS2 protein from Pseudomonas putida MT1, which is a transcriptional activator of the Pm promoter in response to salicylate. The modification allows the response to salicylate. See BBa_K1031911 (original version of this part, which is regulated by benzoate and derivatives of this).
We have inlcuded the sequence of RBS BBa_B0034 by means of the PCR primers in the amplification, as well as the scar that should have been when clonning this part.
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. nasF (see BBa_K1973001) is an attenuator of transcription elongation (terminator) from Klebsiella pneumoniae that decreases the basal expression from the Psal promoter.
We digested BBa_K1973001 as a vector (EcoRI and XbaI) and BBa_K1973033 as an insert (EcoRI and SpeI), and then we ligate them.
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 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.
nasF is an attenuator of transcription elongation (terminator) from Klebsiella pneumoniae that decreases the basal expression from the Psal promoter. The nasR protein disrupts the terminator structure and allows the expression again, providing an useful tool for tight ON/OFF expression systems.
Conversion of the iGEM parts registry to SBOL2.1