B. subtilis genome integration vector, targets pyrD locus
5??? Integration Sequence for the pyrD locus of B. subtilis
The 5??? integration sequence was taken from the B.subtilis chromosome and is homologous to the chromosome from a few hundred bp upstream of the gene's start codon until 300 bp into the gene. The part was synthesised by Geneart.
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 (<bbpart> BBa_K143004</bbpart>) 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 PyrD gene has been a target for numerous integration vectors, including the shuttle vectors pPyr-Cm (GenBank Accession number AY464558) and pPyr-Kan (GenBank Accession number AY464559) 1. Integration into the PyrD locus makes the ''B.subtilis'' auxotrophs for uracil and transformants require about 40ug/ml to allow for growth. This allows us to assay for integration by growing a replica plate with no supplemented uracil to negatively select for transformants.
The PyrD integration sequences were designed from the PyrD gene's Genbank entry#1 and identification of the sequence directly upstream of the gene on the chromosome (found using NCBI's sequence viewer). The upstream and PyrD gene sequence was analysed for restriction sites and primers (with biobrick prefix and suffix sequences) for two approximately equally sized integration sequences were desgined.
3??? Integration Sequence for the pyrD locus of B. subtilis
The 3??? integration sequence was taken from the B.subtilis chromosome and is homologous to the back section of the PyrD gene. It was synthesised by Geneart.
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 3' integration sequence can be added to the back of a Biobrick construct and the 5' integration sequence specific for this locus (BBa_K143008) to the front of the Biobrick construct to allow integration of the Biobrick construct into the chromosome of the gram positive bacterium B.subtilis. The PyrD gene has been a target for numerous integration vectors, including the shuttle vectors pPyr-Cm (GenBank Accession number AY464558) and pPyr-Kan (GenBank Accession number AY464559) [1]. Integration into the PyrD locus makes the B.subtilis auxotrophs for uracil and transformants require about 40ug/ml to allow for growth. This allows us to assay for integration by growing a replica plate with no supplemented uracil to negatively select for transformants.
The PyrD integration sequences was designed from the PyrD gene's Genbank entry[1] and identification of the sequence directly upstream of the gene on the chromosome (found using NCBI's sequence viewer). The upstream and EpsE gene sequence was analysed for restriction sites and primers (with biobrick prefix and suffix sequences) for two approximately equally sized integration sequences were desgined.
Promoter Pveg and RBS spoVG for B. subtilis
Constitutive promoter veg(<bbpart>BBa_K143012</bbpart>) coupled to the strong Ribosome Binding Site spoVG(<bbpart>BBa_K143021</bbpart>) from ''B. subtilis''. Pveg-spoVG can be used in the context of a '''Ribosomes per second''' (RiPS) 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-spoVG were obtained from papers2 and the sequence synthesised by GeneArt
Spectinomycin Resistance Protein (Aad9) - Terminator
Aad9 was PCR cloned from the ''B. subtilis'' integration vector utilising Pfu DNA polymerase and cloned into a BioBrick iwth the double terminator was taken from the registry
Aad9 spectinomycin resistance protein(<bbpart>BBa_K143031</bbpart>) coupled to the double terminator (<bbpart>BBa_B0015</bbpart>). Aad9 confers resistance to spectinomycin. 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 Spectinomycin resistance gene to be incorporated into a closed transcriptional unit.
Aad9 was PCR cloned from the ''B. subtilis'' integration vector utilising Pfu DNA polymerase. 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.
Aad9 was PCR cloned from the ''B. subtilis'' integration vector pDR111 using Pfu DNA polymerase
Aad9 is the spectinomycin resistance gene from ''Enterococcus faecalis''#1. Expression in a host confers resistance to spectinomycin at a concentration of 100μg/μl and has been used in a variety of vectors for both ''B. subtilis'' and ''E. coli'' including pDP870#2, pCOMT-Kan#3 and pIEF16s#4 ====References==== <biblio> #1 pmid=1659306 #2 pmid=16997985 #3 pmid=15060042 #4 pmid=16714443 </biblio>
The sequence of ''B. subtilis'' integration vector pDR111 was searched for the spectinomycin resistance gene and the Biobrick standard applied to the gene sequence upon PCR cloning
SpoVG ribosome binding site (RBS) for B. subtilis
The sequence was taken from a previous research paper [1] and was constructed by Geneart.
Description: SpoVG is an endogenous ribosome binding site from B.subtilis. The sequence of the spoVG ribosome binding site is AAAGGUGGUGA which is complementary to the sequence UUUCCUCCACU 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 -19kcal 1
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.
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