T7 amber expression system in Bacillus subtilis.
Double terminator consisting of BBa_B0010 and BBa_B0012
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
T7 RNA polymerase with tyrosine amber codons
quick change mutagenesis on T7 (BBa_K145001)
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.
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.
Conversion of the iGEM parts registry to SBOL2.1