5??? Integration Sequence for the amyE locus of B. subtilis
The 5' integration sequence can be added to the front of a Biobrick construct and the 3' integration sequence specific for this locus (Part BBa_K143002) to the rear of the Biobrick construct to allow integration of the Biobrick construct into the chromosome of the gram positive bacterium B.subtilis. The AmyE locus was the first locus used for integration into ''B.subtilis'' by Shimotsu and Henner#1 and is still commonly used in vectors such as pDR111#2, pDL#3 and their derivatives. Integration at the AmyE locus removes the ability of ''B.subtilis'' to break down starch, which can be assayed with iodine as described by Cutting and Vander-horn#4. The 5' and 3' integration sequences for the AmyE locus were used to integrate the Imperial 2008 iGEM project primary construct into the ''B.sutbilis'' chromosome. <biblio> #1 pmid=3019840 #2 pmid=14597697 #3 ''Bacillus'' Genetic Stock Center [www.bgsc.org] #4 Cutting, S M.; Vander-Horn, P B. Genetic analysis. In: Harwood C R, Cutting S M. , editors. Molecular biological methods for Bacillus. Chichester, England: John Wiley & Sons, Ltd.; 1990. pp. 27???74. </biblio>
The AmyE integration sequence was taken from the vector after comparison by BLAST to the ''B.subtilis'' chromosome to identify the homologous sequences. The sequence present in both the host chromosome and the plasmid at the 5' end of the gene is the 5' sequence required for integration.
The 5??? integration sequence was taken from the shuttle vector pDR111 which has been used in many studies on ''B.subtilis'', in particular in the studies of transcriptional control#1 #2 #3 <biblio> #1 pmid=14597697 #2 pmid=15937167 #3 pmid=12169614 </biblio>
Low to medium copy BioBrick standard vector
Erik Gullberg, Antonio Ascue Avalos, Erik Lundin
BBa_K592200 is a BioBrick standard vector with low to medium copy p15A replication origin (BBa_I50032) and spectinomycin antibiotic resistance marker, usable for Lambda Red recombineering in E coli. The E coli His operon terminator BBa_B0053 has been replaced with the late terminator of the Salmonella phage P22.
To make the plasmid usable for Lambda Red recombineering, all sequences with homologies to the E coli chromosome had to be removed. To accomplish this, the E coli His operon terminator BBa_B0053 has been replaced with the late terminator of the Salmonella phage P22.
p15A low to medium copy replication origin BBa_I50032
3??? Integration Sequence for the amyE locus of B. subtilis
Integration sequences allow DNA to be incorporated into the chromosome of a host cell at a specific locus using leading (5') and trailing (3') DNA sequences that are the same as those at a specific locus of the chromosome. The 5' integration sequence can be added to the front of a Biobrick construct and the 3' integration sequence specific for this locus (Part BBa_K143002) to the rear of the Biobrick construct to allow integration of the Biobrick construct into the chromosome of the gram positive bacterium B.subtilis. The AmyE locus was the first locus used for integration into B.subtilis by Shimotsu and Henner[1] and is still commonly used in vectors such as pDR111[2], pDL[3] and their derivatives. Integration at the AmyE locus removes the ability of B.subtilis to break down starch, which can be assayed with iodine as described by Cutting and Vander-horn[4]. The 5' and 3' integration sequences for the AmyE locus were used to integrate the Imperial 2008 iGEM project primary construct into the B.sutbilis chromosome. References 1. Shimotsu H and Henner DJ. Construction of a single-copy integration vector and its use in analysis of regulation of the trp operon of Bacillus subtilis. Gene 1986; 43(1-2) 85-94. pmid:3019840 2.Nakano S, Kster-Schck E, Grossman AD, and Zuber P. Spx-dependent global transcriptional control is induced by thiol-specific oxidative stress in Bacillus subtilis. Proc Natl Acad Sci U S A 2003 Nov 11; 100(23) 13603-8. doi:10.1073/pnas.2235180100 pmid:14597697 3.Bacillus Genetic Stock Center; www.bgsc.org 4.Cutting, S M.; Vander-Horn, P B. Genetic analysis. In: Harwood C R, Cutting S M. , editors. Molecular biological methods for Bacillus. Chichester, England: John Wiley & Sons, Ltd.; 1990. pp. 27???74.
The AmyE integration sequence was taken from the vector after comparison by BLAST to the B.subtilis chromosome to identify the homologous sequences. The sequence present in both the host chromosome and the plasmid at the 3' end of the gene is the 3' sequence required for integration
The 3??? integration sequence was taken from the shuttle vector pDR111 which has been used in many studies on B.subtilis, in particular in the studies of transcriptional control[1,2,3] References 1.Shimotsu H and Henner DJ. Construction of a single-copy integration vector and its use in analysis of regulation of the trp operon of Bacillus subtilis. Gene 1986; 43(1-2) 85-94. pmid:3019840. 2.Erwin KN, Nakano S, and Zuber P. Sulfate-dependent repression of genes that function in organosulfur metabolism in Bacillus subtilis requires Spx. J Bacteriol 2005 Jun; 187(12) 4042-9. doi:10.1128/JB.187.12.4042-4049.2005 pmid:15937167 3.Britton RA, Eichenberger P, Gonzalez-Pastor JE, Fawcett P, Monson R, Losick R, and Grossman AD. Genome-wide analysis of the stationary-phase sigma factor (sigma-H) regulon of Bacillus subtilis. J Bacteriol 2002 Sep; 184(17) 4881-90. pmid:12169614
Encryption key sequence in integration plasmid (BBa_K823023)
The iGEM team Groningen 2016 worked on bioencryption in order to safely store data in DNA. In our project we were mainly working with two sequences of DNA which were the encrypted message sequence and the encryption key sequence. The key sequence was created by our software. It is therefore artificial DNA. We ordered the encrypted key sequence as gBlock from IDT (Integrated DNA technologies). This part was created in the interest of having a genomic integration plasmid with our key sequence. We used an available BioBrick BBa_K823023 from igem Munich 2012. It is an integration plasmid for Bacillus subtilis, which can be used for cloning in E. coli as well. An RFP is inserted in BBa_K823023 for more efficient screening after transformation.
Key sequence: Created by our CryptoGErM software. http://2016.igem.org/Team:Groningen/Encoding BBa_K823023: Created by iGEM Munich 2012. http://parts.igem.org/Part:BBa_K823023
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
Encrypted message sequence in integration plasmid (BBa_K823023)
The iGEM team Groningen 2016 worked on bioencryption in order to safely store data in DNA. In our project we were mainly working with two sequences of DNA which were the encrypted message sequence and the encryption key sequence. The message sequence was created by our software. It is therefore artificial DNA. We ordered the encrypted message sequence as gBlock from IDT (Integrated DNA technologies). This part was created in the interest of having a genomic integration plasmid with our message sequence. We used an available BioBrick BBa_K823023 from igem Munich 2012. It is an integration plasmid for Bacillus subtilis, which can be used for cloning in E. coli as well. An RFP is inserted in BBa_K823023 for more efficient screening after transformation.
The iGEM team Groningen 2016 worked on bioencryption in order to safely store data in DNA. In our project we were mainly working with two sequences of DNA which were the encrypted message sequence and the encryption key sequence. The message sequence was created by our software. It is therefore artificial DNA. We ordered the encrypted message sequence as gBlock from IDT (Integrated DNA technologies). This part was created in the interest of having a genomic integration plasmid with our message sequence. We used an available BioBrick BBa_K823023 from igem Munich 2012. It is an integration plasmid for Bacillus subtilis, which can be used for cloning in E. coli as well. An RFP is inserted in BBa_K823023 for more efficient screening after transformation. For more information see plasmid construction under our lab journal: http://2016.igem.org/Team:Groningen/Experiments
Message sequence: Created by our CryptoGErM software. http://2016.igem.org/Team:Groningen/Encoding BBa_K823023: Created by iGEM Munich 2012. http://parts.igem.org/Part:BBa_K823023
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.
Low to medium copy Lambda Red recombineering compatible plasmid
BBa_K592202 is a BioBrick friendly vector with low to medium copy p15A replication origin (BBa_I50032) and a kanamycin antibiotic resistance marker, usable for Lambda Red recombineering in E coli. The E coli His operon terminator BBa_B0053 has been replaced with the late terminator of the Salmonella phage P22, and the kanamycin resistance marker is flanked by FRT sites so that the resistance marker can be removed after chromosomal integration using the FLP recombinase.
To make the plasmid usable for Lambda Red recombineering, all sequences with homologies to the E coli chromosome had to be removed. To accomplish this, the E coli His operon terminator BBa_B0053 has been replaced with the late terminator of the Salmonella phage P22. To be able to remove the antibiotic resistance marker after chromosomal integration, the kanamycin marker from the pKD4 plasmid (Datsenko and Wanner, 2000) was used. The resistance marker can be removed after chromosomal integration using the FLP recombinase, leaving only the plasmid insert and a scar behind.
The P22 late terminator come from the genome of the Salmonella phage P22 (GenBank: AF217253.1, 19662..19702), and the kanamycin resistance marker flanked by FRT sites comes from the pKD4 plasmid (Datsenko and Wanner, 2000).