AmyE integratable PoPS generator (P43-gsiB) (with CmR)
The amyE 5' integration sequence was PCR cloned from the ''B. subtilis'' integration vector utilising Pfu DNA polymerase and cloned into a BioBrick with the P43-gsiB that was synthesised by GeneArt and Chloramphenicol adenyltransferase and the double terminator that were obtained from the registry.
AmyE 5' Integration sequence(<bbpart>BBa_K143001</bbpart>) coupled to a chloramphenicol resistance generator in closed transcriptional unit (Parts <bbpart>BBa_K143050</bbpart> and <bbpart>BBa_K143064</bbpart>) and the PoPS generator P43-gsiB (<bbpart>BBa_K143050</bbpart>). The amyE 5' integration sequence allows integration into the ''B. subtilis'' genome at the amyE locus if the 3' amyE integration sequence(<bbpart>BBa_K143002</bbpart>) is cloned onto the 3' end of the construct. The chloramphenicol adenyltransferase give resistance to chloramphenicol while the terminator prevents readthrough. The P43-gsiB promoter and RBS for ''B. subtilis'' constitutively generate a PoPS output.
The amyE 5' integration sequence was PCR cloned from the ''B. subtilis'' integration vector pDR111 utilising Pfu DNA polymerase. The sequence of P43-gsiB was obtained from papers. Chloramphenicol adenyltransferase and the double terminator were obtained from the registry.
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.
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.
Promoter 43 a constitutive promoter for B. subtilis
The part was designed using the sequence from the ''B.subtilis'' genome and from previously published papers 2. This sequence was then synthesised by Geneart.
Promoter 43 is a constitutive promoter that constitutively expresses the P43 protein in ''B.subtilis''. This promoter has been shown to be recognized and active during the exponential and lag phases of growth. It has been hypothesized that the ability to recognize the promoter in exponential and lag phase of growth is due to the recognition of the promoter by both sigma factor 55 (the major sigma factor) and sigma factor 37 (the lag phase sigma factor) 1. The P43 promoter has been previously used for constitutive expression of exogenous genes within ''B.subtilis'' vectors 2. The context with which we used the promoter P43 is as a '''Polymerase Per Second''' (PoPS) generator.
The biobrick part was designed to include the binding sites for both the sigma factor A and B. In addition the biobrick standard was applied to the promoter P43 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.
Conversion of the iGEM parts registry to SBOL2.1