B.subtilis transformation vector, targets amyE locus
Existing biobricks, <bbpart>BBa_K143070</bbpart>, <bbpart>BBa_K316002</bbpart>, <bbpart>BBa_K316014</bbpart> <bbpart>BBa_K143002</bbpart>
This vector has been designed using the amyE 5' and 3' integration sequences for integration into B.subtilis genome '''AmyE locus''' This vector has been designed using the amyE 5' <bbpart>BBa_K143008</bbpart> and 3 <bbpart>BBa_K143009</bbpart>' integration sequences for integration into ''B. subtilis'' genome. Insertion into the amyE locus provides a selection marker as the bacterium will no longer be able to breakdown starch. An iodine assay can be used to confirm integration. This phenotype makes the transformed bacterium considerably less likely to survive in natural environments. '''Chloramphenicol Resistance''' This vector also contains a positive selection marker, flanked by two dif sites. Chloramphenicol acetyltransferase <bbpart>BBa_J31005</bbpart> provides resistance to chloramphenicol antibiotic. Dif <bbpart>BBa_K316002</bbpart> sites allow excision of the antibiotic marker after integration, thus allowing the same marker to be used again or as a precaution against horizontal gene transfer. '''Blunt end cloning site''' PmeI restriction site <bbpart>BBa_K316013</bbpart> is designed as a cloning site. Due to the 8bp recognition sequence it is a rare site that can be used to cut the vector only once. Please see ???Part Design??? section for design considerations and parts used.
Standard biobrick assembly [http://partsregistry.org/Assembly:Standard_assembly]
5??? Integration Sequence for the amyE locus of B. subtilis
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>
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.
3??? Integration Sequence for the amyE locus of B. subtilis
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
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
Promoter Pveg and RBS gsiB for B. subtilis
Constitutive promoter veg(<bbpart>BBa_K143012</bbpart>) coupled to the strong Ribosome Binding Site gsiB(<bbpart>BBa_K143020</bbpart>) from ''B. subtilis''. Pveg-gsiB can be used in the context of a Polymerases per second (PoPS) output generator '''To get the highest level of translation from this Promoter-RBS combination it must be connected to a coding region preceded by a coding region prefix1. A standard prefix will increase the distance between the RBS and the start codon, reducing translational efficiency.'''
The sequence of Pveg was obtained from the DBTBS1 and RBS-gsiB were obtained from papers2 and the sequence synthesised by GeneArt
Chloramphenicol resistance protein - Terminator
The Chloraphemicol acetyltransferase and double terminator were taken both taken from the registry.
Chloraphemicol acetyltransferase protein(<bbpart>BBa_J31005</bbpart>) coupled to the double terminator (<bbpart>BBa_B0015</bbpart>). Chloraphemicol acetyltransferase confers resistance to Chloraphemicol. The double terminator is the most commonly used terminator and is a combination of parts <bbpart>BBa_B0010</bbpart> and <bbpart>BBa_B0012</bbpart>. The double terminator allows the CAT to be incorporated into a closed transcriptional unit.
Chloraphemicol acetyltransferase is an exisiting registry protein. The double terminator is the most commonly used registry termiantor.
The dif sites were made by annealing synthestised oligoes.
Dif sites are naturally found in B.subtilis and are used by this organism during genome replication.
The dif site was made by oligos designed to make overhangs for EcoRI and SpeI ( and ) or XbaI and PstI ( and ) to be used in standard Biobrick or 3A cloning.
Dif sequence followed by PmeI recognition site
Oligonucleotide synthesis of single stranded primers.
This composite part of <bbpart>BBa_K143000</bbpart> and <bbpart>BBa_K143013</bbpart>. The dif site can be used in conjunction with another dif site in another part of the vector to remove a sequence between the two dif sites. PmeI site can be used for blunt end cloning of a DNA sequence behind the dif site. Please see ???Part Design??? section for design considerations and parts used.
This part was designed to be cloned using standard biobrick methods. Two single stranded, synthetic oligos were annealed to produce a double stranded DNA sequence with single stranded overhangs identical to the product of digestion by EcoRI and SpeI. Thus compatible with biobrick cloning methods.
Derived from the TE terminator of T7 bacteriophage between Genes 1.3 and 1.4 <genbank>V01146</genbank>.
Transcription terminator for the E.coli RNA polymerase.
Suggested by Sri Kosuri and Drew Endy as a high efficiency terminator. The 5' end cutoff was placed immediately after the TAA stop codon and the 3' end cutoff was placed just prior to the RBS of Gene 1.4 (before AAGGAG). Use anywhere transcription should be stopped when the gene of interest is upstream of this terminator.
Transcriptional terminator consisting of a 64 bp stem-loop.
Promoter veg a constitutive promoter for B. subtilis
The Pveg promoter was suggested to us by Dr. Jan-Willem Veening of Newcastle University. This sequence supplied was compared to that of the DBTBS database#3 then a section containing the binding site synthesised by Geneart.
Pveg is a constitutive promoter that constitutively expresses the P43 protein in ''B.subtilis''. Pveg contains binding sites for the ''B.sutbilis'' major sigma factor#1. Pveg in ''B.subtilis'' utilises two binding sites to cause high expression of genes#2, however our Pveg is lacking the upstream site to give a medium level of gene expression. It has been noted that the sporulation master regulatoion factor spoOA interacts with Pveg though it is not known how#3. The context with which we used the promoter Pveg is as a '''Polymerase Per Second''' (PoPS) generator.
The biobrick part was designed to include a single binding site for the ''B.subtilis major sigma factor. In addition the biobrick standard was applied to the promoter Pveg sequence.
GsiB ribosome binding site (RBS) for B. subtilis
The sequence was taken from a previous research paper [1] and was constructed by Geneart
GsiB is an endogenous ribosome binding site from ''B.subtilis''. The sequence of the gsiB ribosome binding site is '''AAAGGAGG''' which is complementary to the sequence '''UUUCCUCC''' from the 3' region of the 16s rRNA from ''B.subtilis''. GsiB is an endogenous ribosome binding site (RBS) from ''B.subtilis''. The sequence of the gsiB ribosome binding site is '''AAAGGAGG''' which is complementary to the sequence '''UUUCCUCC''' from the 3' region of the 16s rRNA from ''B.subtilis''. Previous research showed that the predicted binding energy of the 16s rRNA to the RBS is -9.3kcal.
In order to ensure that the RBS is functional the actual ribosome binding site was maintained and the distance between the RBS and the start codon maintained. In order to conform to the biobrick standard the sequence flanking the RBS had to be changed but the distance between the promoter and RBS, and start codon and RBS was maintained.
chloramphenicol acetyltransferase (forwards, CmF) [cf. BBa_J31004]
When a promoter and an RBS are in front of the gene, the cell will express Chloramphenicol resistance. Because it contains full biobrick ends, this part can be used to easily add chloramphenicol resistance to any part without changing plasmid vectors.
8bp recognition sequence for PmeI restriction endonuclease
Information about PmeI restriction endonuclease is available at [http://www.neb.com/nebecomm/products/productR0560.asp]. The recognition site is a 8bp sequence GTTTAAAC. Pme produces a blunt cut after GTTT.
Conversion of the iGEM parts registry to SBOL2.1