B. subtilis Episomal Vector with Constitutive GFP
See data for BBa_K90401. This part is the same, but with the pUpp promoter, a gram-positive RBS, and a GFP with terminator.
This episomal vector has within its BioBrick region a constitutively expressed GFP gene. It uses the pUpp strong constitutive promoter and a consensus Bacillus RBS sequence.
To build this part, we used 4 different linear DNA pieces assembled with 15 base pair homologous regions PCRed to the ends. These 4 different pieces were then assembled via the InFusion assembly method by Clontech.
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.
green fluorescent protein derived from jellyfish Aequeora victoria wild-type GFP (SwissProt: P42212
GFP (mut3b) [note that this part does not have a barcode]
Gram-Positive Strong Constitutive Promoter
We PCRed this part out of the pAD43-25 gram positive vector, acquired from the Bacillus Genetic Stock Center (bgsc.org), catalog number ECE166.
This is a strong constitutive (always-on) promoter for use with gram-positive organisms. It requires a downstream gram-positive ribosomal binding site and coding sequence to function. We only have experience using this part in B. subtilis, but it should function in most gram-positive organisms. It has not been tested and will most likely not work in E. coli. The promoter comes from the cis-regulatory region of the uracil phosphatase in Bacillus cereus. We functionally annotated the promoter ourselves using the DBTBS database motif search feature. (dbtbs.hgc.jp)
This part generates high expression in gram-positive organisms, and it may be possible to generate deleterious effects on the cell if used to express certain protein products.
Gram-positive Shuttle Vector for Episomal Expression
This part was made via InFusion assembly from many separate pieces. The backbone comes from PAD123 (Bacillus Stock Center catalog number ECE166), and contains replication origins for B. subtilis and E. coli, as well as resistance markers for Ampicillin and Chloramphenicol.
This episomal plasmid is capable of transformation into both Gram-negative and Gram-positive organisms. It replicates as an episomal plasmid and does not integrate into the genome. For selection in E. coli, use Ampicillin, 100 ??g per mL. For selection in B. subtilis (and other similar gram-positive organisms), use Chloramphenicol, 5 ??g per mL (not 35).
We had to remove a PstI site inside the coding region of the beta-lactamase gene coding for Ampicillin resistance, and thus the backbone had to be PCRed from two parts, with a joining primer filling in cut site with a degenerate codon.
This is the consensus RBS for Bacillus subtilis.
The part must be 8 nucleotides away from the first methionine of the coding sequence, and so we added 'TGT' downstream of the actual binding sequence: AAAGGAGG. This, combined with the truncated non-CDS BioBrick prefix scar, gives exactly 8 'spacer bases' between the 3' end of the RBS and the start of the coding sequence.
Conversion of the iGEM parts registry to SBOL2.1