Plasmid for Chromosomal Integration in Yeast at His3
This part is designed to allow simple chromosomal integration of BioBrick plasmids. It is based off of the bacterial pSB1AT3 backbone allowing for normal DNA manipulation to occur in bacteria. However the BioBrick cut sites are flanked by homologous regions to the His3 locus in S. cerevisiae. To transform S. cerevisiae the plasmid is cut with BbsI creating two non-sticky ends. The linear fragment then contains His3 homologous sequences at its ends that can be used to chromosomally integrate the BioBrick plasmid into the yeast genome. Transformants can then be selected for using His- medium.
This part was derived from plasmid pSB1AT3. Two successive rounds of PCR inserted a BbsI cut site and 40 bp homologous region in before the prefix and after the suffix. The following Primers were used: Forward 1 GCACAGAACAATAACCTGCTGGAAACGAAGATAAATCgaagacGATTACTTCGCGTTATGCAGGC Reverse 1 GCATCTTCTCAAATATGCTTCCCAGCCTGCTTATCcttctgAAATTCTGCCTCGTGATACGCC Forward 2 tactagtagcggccgctgcagTCTTAACCCAACTGCACAGAACAATAACCTGCTGGAAACG Reverse 2 ctctagaagcggccgcgaattcTTAGTATTGCTGGCCGCATCTTCTCAAATATGCTTCCCAGCC
pORE Gus Reporter Series Vector with BiOBrick MCS (Kan resistance)
Vector for transformation of plants. Cloning in E. coli, transfer to Agrobacterium, integrates into plant genome. Kan resistance marker in bacteria, confers Basta resistance in plants. Has a constitutive plant promoter and in-frame Gus reporter. Conforms to Silver BioBrick Assembly Standard 21.
Modified vector pORE_R from the Arabidopsis Information Resource
2-oxoglutarate oxygenase/decarboxylase is an Ethylene Forming Enzyme (EFE) that catalyze Ethylene biosynthesis from 2-oxoglutarate. This part is in the pXyl vector that has been sent to the Igem UNITN 2013 team from the LMU Munich 2012 team. This part was cloned by the iGEM Trento 2013 team for the creation of an aerobically engineered pathway for the control of fruit ripening. Further information about this part and its characterization can be found in the iGEM Trento 2013 wiki.
===Design Notes=== The coding sequence was taken from Pseudomonas syringae pv. phaseolicola PK2. We firstly optimized the codons for both B. subtilis and E. coli and then synthesized the gene with an RBS sequence upstream of the start codon. We decided to include in the CDS two restriction sites (NgoMIV and AgeI) in order to allow people to use the Freiburg assembly. GAATTCGCGGCCGCTTCTAGATAAGGAGGAACTACTATGGCCGGCACC... ...TCGACCGGTTAATACTAGTAGCGGCCGCTGCAG
This enzyme was firstly purified from Pseudomonas Siringae pv. phaseolicola PK2, a 2-oxoglutarate-dependent ethylene producing bacterium [1]. The CDS has been optimized for both E.coli and B.subtilis. We decided to include an RBS sequence too.
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. lapG (see BBa_K1973000) 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).
Designs are described in basic parts pages
Basic parts were cloned into the miniTn7BB-Gm Backbone
Thomas Dohmen, Milana Kremenovic, Yannick Kristiansen, Tim Maier
pBS1C: Bacillus subtilis vector, amyE integration, CAM-resistance
This vector integrates into the amyE-site of Bacillus subtilis via double recombination. The two recombination sites flank a standard BioBrick multiple cloning site (with RFP-cassette), as well as a chloramphenicol resistance cassette (5 ??g/ml). The backbone does not contain the restriction sites EcoRI, XbaI, SpeI, PstI, NgoMIV nor AgeI. The vector is replicative in Escherichia coli and provides resistance to ampicillin (100 ??g/ml). Before transformation into B. subtilis, this vector should be linearized with ScaI.
This is one of three empty vectors with compatible integration loci and resistance cassettes to allow combination of up to three constructs of choice in one single strain.
pSBBs1C: Empty backbone for integration into Bacillus subtilis amyE locus
This part is an empty backbone vector for the usage in Bacillus subtilis. It integrates in the amyE locus and can be selected with Chloramphenicol (cat gene). It has a ampicillin resistance for cloning in E.coli. It originates from the Bacillus vector pDG1662. This BioBrick is part of the LMU 2012 Bacillus BioBrick Box project: http://2012.igem.org/Team:LMU-Munich/Bacillus_BioBricks
We removed any forbidden restriction sites by site-directed mutagenesis PCR.
This backbone is an igem compatible version of the Bacillus vector pDG1662
contains restriction sites needed for assembly of BioBricks
pBS2E: Bacillus subtilis vector, lacA integration, MLS resistance
This part is an empty backbone vector for the usage in Bacillus subtilis. It integrates in the lacA locus and can be selected with erythromycin + lincomycin (MLS). It has a ampicillin resistance for cloning in E.coli.
Compatible with pBs1C and pBs4S in one strain.
This backbone is a BioBricked version of the B. subtilis vector pAX01. This BioBrick is part of the LMU 2012 igem project [http://2012.igem.org/Team:LMU-Munich/Bacillus_BioBricks '''''Bacillus''B'''io'''B'''rick'''B'''ox] Reference: [http://www.ncbi.nlm.nih.gov/pubmed/11274134 H??rtl ''et al.'']
pSBBs4S-Pxyl: Integrative expression vector for Bacillus subtilis</i
This part is an expression vector for Bacillus subtilis. It integrates at the thrC locus. It has a ampicillin resistance for cloning in E.coli and chloramphenicol resistance for selection in B.subtilis. The multiple cloning site downstream of a Pxyl promoter, that is inducible with xylose. This BioBrick is part of the LMU 2012 igem project Bacillus BioBrick Box http://2012.igem.org/Team:LMU-Munich/Bacillus_BioBricks
We removed any forbidden restriction sites by site-directed mutagenesis PCR. The clonging site also includes a rfp for selection for cloning in E.coli.
This part is an igem compatible version of the Bacillus expression vector pXT
The miniTn7BB-Cm minitransposon 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 maintain the mini-Tn7-Cm 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-Cm 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 chloramphenicol resistance cassette (Cm) 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.
NcoI and SphI sites were added at the ends of the chloramphenicol resistance cassette to facilitate cloning
This construct is the result of replacing the gentamycin resistance cassette in pUC18SfiI-miniTn7BB-Gm by a chloramphenicol resistance cassette obtained from pBS1C3 by PCR amplification
pSBBs2E: Empty backbone for integration into Bacillus subtilis lacA locus
This part is an empty backbone vector for the usage in Bacillus subtilis. It integrates in the lacA locus and can be selected with erythromycin + lincomycin (MLS). It has a ampicillin resistance for cloning in E.coli. This BioBrick is part of the LMU 2012 Bacillus BioBrick Box project: http://2012.igem.org/Team:LMU-Munich/Bacillus_BioBricks
We removed any forbidden restriction sites by site-directed mutagenesis PCR. The clonging site also includes a rfp for selection in E.coli.
This part is an igem compatible version of the Bacillus subtilis vector pAX01
pORE Open Series Vector with BioBrick MCS (Basta resistance)
Vector for transformation of plants. Cloning in E. coli, transfer to Agrobacterium, integrates into plant genome. Kan resistance marker in bacteria, confers Basta resistance in plants. Has no promoter or reporter.
Modified vector pORE_O1 from the Arabidopsis Information Resource
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.
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.
pORE GFP Reporter Series Vector with BioBrick MCS (Kan resistance)
Vector for transformation of plants. Cloning in E. coli, transfer to Agrobacterium, integrates into plant genome. Kan resistance marker in bacteria, confers Kan resistance in plants. Has constitutive plant promoter and in-frame GFP reporter. Conforms to Silver BIoBrick Assembly Standard 21.
Modified vector pORE_R2 from the Arabidopsis Information Resource
This is a targeting vector for homologous recombination into the ADE4 locus of S. cerevisiae. This vector can be used in cloning free cloning (ref. 2010 uOttawa iGEM wiki). Yeast strains with an ADE1 or ADE2 deletion or mutation have red colonies; by integrating the constructs using our protocol the ADE4 gene is knocked out and the colonies express a white phenotype.
pSBBs1C-lacZ (lacZ reporter vector for B. subtilis)
This part is a backbone vector for Bacillus subtilis. It integrates at the amyE locus. It has a Ampicillin resistance for cloning in E.coli and Chloramphenicol resistance for B.subtilis. The multiple cloning site is followed by a lacZ gene for reporter assays. This BioBrick is part of the LMU 2012 igem project Bacillus BioBrick Box http://2012.igem.org/Team:LMU-Munich/Bacillus_BioBricks
This reporter vector can be used with the normal BioBrick assembly strategies.
This vector is the Bacillus vector pAC6 brought into the iGEM BioBrick standard.
Plasmid for Chromosomal Integration in Yeast at Ura3
This part is designed to allow simple chromosomal integration of BioBrick plasmids. It is based off of the bacterial pSB1AT3 backbone allowing for normal DNA manipulation to occur in bacteria. However the BioBrick cut sites are flanked by homologous regions to the Ura3 locus in S. cerevisiae. To transform S. cerevisiae the plasmid is cut with BbsI creating two non-sticky ends. The linear fragment then contains Ura3 homologous sequences at its ends that can be used to chromosomally integrate the BioBrick plasmid into the yeast genome. Transformants can then be selected for using Ura- medium.
Ciprofloxacin resistance cassette in integration plasmid (BBa_K823023)
This part is a ciprofloxacin resistance cassette. It is containing a PAtpI constitutive promoter, ciprofloxacin resistance gene qnrS1 and double terminator. The promoter PAtpI ([http://parts.igem.org/Part:BBa_K1930005 ''BBa_K1930005'']) is a constitutive promoter which has its origin in Bacillus subtilis. It is responsible for the expression of atpA gene (ATP synthesis) during the first 30 minutes of the germination of B. subtilis. atpA gene is part of an operon (atpI-atpB-atpE-atpF-atpH-atpA-atpG-atpD-atpC), therefore the promoter region in front of the first protein coding gene (atpI) in this operon was chosen. Ciprofloxacin resistance gene qnrS1 is found naturally in E. coli, and other Gram-negative strains. qnr genes code for pentapeptide repeat proteins.??These proteins reduce susceptibility to quinolones by protecting the complex of DNA and DNA gyrase enzyme from the inhibitory effect of quinolones. Double terminator ([http://parts.igem.org/Part:BBa_B0015 ''BBa_B0015'']) is the most commonly used terminator. For more information see here: http://parts.igem.org/Part:BBa_B0015.
Ciprofloxacin resistance cassette: Ordered as gBlock. IDT (Integrated DNA technologies). BBa_K823023: Created by iGEM Munich 2012. http://parts.igem.org/Part:BBa_K823023.
pORE Open Serices Vector with BioBrick MCS (Kan resistance)
Vector for transformation of plants. Cloning in E. coli, transfer to Agrobacterium, integrates into plant genome. Kan resistance marker in bacteria, confers Kan resistance in plants. Has no promoter or reporter. Conforms to Silver BioBrick Assembly Standard 21.
pSBBs4S: Empty backbone for integration into Bacillus subtilis thrC locus
This part is an empty backbone vector for the usage in Bacillus subtilis. It integrates in the thrC locus and can be selected with Spectinomycin cassette. It has a ampicillin resistance for cloning in E.coli. It originates from the Bacillus vector pDG1731.
This plasmid can be used as empty backbone vectors like pSB1C3 but is able to integrate in B.subtilis. The clonging site also includes a rfp for selection.
pSBBs3C-luxABCDE (lux reporter vector for B.subtilis)
This part is a reporter vector for Bacillus subtilis. It integrates at the sacA locus of B.subtilis genome. It has a ampicillin resistance for cloning in E.coli and Chloramphenicol resistance for selection in B. subtilis. The multiple cloning site has a rfp for selection and is followed by the luxABCDE gene cluster for luminescence reporter assays. This BioBrick is part of the LMU-Munich 2012 igem project Bacillus BioBrickBOX http://2012.igem.org/Team:LMU-Munich/Bacillus_BioBricks
This vector is an igem compatible version of the pAH328 Bacillus reporter vecotr.
pBs4S: Bacillus subtilis vector, thrC integration, spec resistance
This part is an empty backbone vector for the usage in Bacillus subtilis. It integrates in the thrC locus and can be selected with Spectinomycin cassette. It has a ampicillin resistance for cloning in E.coli. This backbone is a BioBricked version of the B. subtilis vector pDG1731.
This vector is compatible with pBS1C and pBS2E in one strain.
This BioBrick is part of the LMU 2012 igem project BacillusBioBrickBox For handling of B. subtilis vectors, please see here. The transformation into B. subtilis is explained here.
Gram-positive Shuttle Vector for Chromosomal Integration
This is pBSINT1, a BioBrick-compatible Shuttle Vector that allows for single-copy chromosomal insertion into Bacillus subtilis. It contains the replication origin and Biobrick cut site from pSB1A3 for normal replication in E. coli. It also holds the 5' and 3' flanking regions of the AmyE gene in Bacillus subtilis, and uses these regions of homology to integrate and replace the contents of that locus on the chromosome via a double crossover event. It has two selectable resistance markers, one for Chloramphenicol (5??g/ml) in Bacillus subtiis, and one for Ampicillin (100 ??g/ml) in E. coli. This vector can be used to construct large BioBrick parts using traditional gram-negative-based transformation and minipreps, and then also be used to integrate the completed part into the genome of a gram-positive chassis. As the figure explains, this vector was made via InFusion assembly from several pieces at once. For a detailed protocol of transformation in Bacillus and how to detect integration events at the AmyE locus, see the Cambridge iGEM 2008 wiki protocols page: [http://2008.igem.org/Team:Cambridge/Bacillus_subtilis_transformation]
We attempted to design the PCR ends to not interfere with the AmyE flanking regions, and also to have the same annealing temperatures. To see the primers we used, see our Primer page under the Bacillus subproject: [http://2008.igem.org/Team:Cambridge/Bacillus/Primers]
As explained in the image, this vector was assembled at once from 4 separate pieces. The green and red regions (biobrick cut site and E. coli replication/Amp resistance) come from PBS1A3. The blue pieces are the flanking AmyE regions from ECE112, which was obtained via the Bacillus Genetic Stock Center.
amyE back; partial alpha-amylase; similar to Genbank U46196
3' UTR site of alcohol oxidase 1 gene (aox1)
3' UTR site of alcohol oxidase 1 gene (AOX1) from K. pastoris used as a recombination site.
The DNA sequence from plasmid pPIC9K of Invitrogen (http://products.invitrogen.com/ivgn/product/V17520?ICID==%3Dsearch-product) was used for primer design. But the genomic DNA of Pichia pastoris wildtype X33 was used for amplification via PCR.