this part was designed with gram positive bacteria in mind. However, we are unable to characterize it in gram positive organism in E. coli
We have to find a promoter that can be reversibly activated by a protein
**highly** engineered mutant of red fluorescent protein from Discosoma striata (coral)
Campbell et al., PNAS v99 p7877 URL
monomeric RFP: Red Fluorescent Protein. Excitation peak: 584 nm Emission peak: 607 nm
TAATAA double stop codon added (DE). Four silent mutations made to remove three EcoRI sites and one PstI site: A28G, A76G, A349G, G337A.
green fluorescent protein derived from jellyfish Aequeora victoria wild-type GFP (SwissProt: P42212
GFP (mut3b) [note that this part does not have a barcode]
Double terminator consisting of BBa_B0010 and BBa_B0012
The part was designed using the sequence from the ''B.subtilis'' genome and from previously published papers 12. This sequence was then synthesised by Geneart.
Promoter Xylose is an inducible promoter that has been designed for high expression in ''B.subtilis''. Gene expression under the promoter xylose can be induced by addition of xylose. The context with which we used the promoter xylose, was to take an input of xylose and give '''Polymerase Per Second'''(PoPS) as an output. Promoter xylose is an inducible promoter that has been designed for high expression in ''B.subtilis''. Gene expression under the promoter xylose can be induced by addition of xylose. The context with which we used the promoter xylose, was to take an input of xylose and give '''Polymerase Per Second'''(PoPS) as an output. Xylose does not induce the promoter xylose directly, but requires the transcriptional regulator '''XylR''', (<bbpart>BBa_K143036</bbpart>) This means that XylR must be constitutively expressed in ''B.subtilis'' in order to use the promoter xylose.
Biobrick standard was applied to the promoter xylose sequence.
LacI (Lva-, N-terminal deletion) regulatory protein
The LacI gene was cloned from''B. subtilis'' shuttle vector pDR111 using Pfu DNA polymerase PCR
LacI is a regulatory protein responsible for the repression of many catabolite genes. Transcription is regulated by proteins which bind operator sequences around the transcription start site. These proteins can positively affect transcription (activators) or negatively affect transcription (reppresors). Some repressor proteins can be inactivted however by addition of an inducer, such as IPTG or certain sugars. LacI if the regulator protein for the lactose operon in ''E.coli'' and the hyper-spank protein of ''B. subtilis''#1(<bbpart>BBaK143015</bbpart>) and is responsible for ensuring that in the absence of lactose (or IPTG) that there is no expression trough these promoter. LacI is not endogenous to ''B. subtilis'', so LacI will need to be expressed in the host in order for the hyper-spank promoter to be regulated. In the presence of IPTG or lactose, the LacI tetramer is unable to bind DNA and so transcription resumes. This version of LacI lacks a Lva degradation tag and has a small(3 amino acid) N-terminal deletion relative to the current registry LacI (<bbpart>BBa_C0012</bbpart)> and is derivatives. The N-terminal deletion appears to be common to most of the LacI genes used in conjunction with ''B. subtilis'' though both forms are found in ''E.coli'' (in differing strains). LacI was used in conjunction with the '''Hyper-spank promoter''' (<bbpart>BBa_K143015<bbpart>) and acted as an input adaptor for a '''Polymerases per second''' (POPS) output ====References==== <biblio> #1 pmid=16166525 </biblio>
LacI was located in the sequence of the ''B. subtilis'' shuttle vector pDR111. This version of LacI lacks a Ltva degradation sequence and has a small N-terminal deletion that is observed in many LacI used in studies on ''B.subtilis''. In particular, this LacI protein is used in pDR111 to regulate expression of the inducible Phyper-spank protein (<bbpart>BBa_K143015</bbpart>) (also used in the pDR111 vector). The BioBrick prefix and suffix were applied to the gene
The XylR protein was PCR cloned form the ''B. subtilis'' genome using Pfu DNA polymerase
Transcription is regulated by proteins which bind operator sequences around the transcription start site. These proteins can positively affect transcription (activators) or negatively affect transcription (reppresors). Some repressor proteins can be inactivted however by addition of an inducer, such as xylose. XylR if the regulator protein for the Xylose operon in ''B. subtilis''#1 and is responsible for ensuring that in the absence of xylose the xylose metabolism proteins are not expressed. Though endogenous to ''B. subtilis'', to minimise the leakage of a xylose inducible promoter XylR should be over-expressed. In the presence of xylose, the XylR tetramer is unable to bind DNA and so transcription resumes. It must be noted that in all ''B. subtilis'' strains that do not have the Xylose operon knocked out '''the xylose inducer will gradually be metabolised by the host''' XylR was used in conjunction with the '''Xylose operon promoter''' (<bbpart>BBa_K143014<bbpart>) and acted as an input adaptor for a '''Polymerases per second''' (POPS) output
The XylR protein was identified in the genome using its Genbank entry#2 and NCBI's sequence viewer and PCR primers designed from the sequence. Biobrick prefix and suffix sequences were added and the gene cloned by PCR with Pfu DNA polymerase
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.
The part was designed using the sequence from the ''B.subtilis'' genome and from previously published papers 123. This sequence was then synthesised by Geneart.
Promoter hyper-spank is an inducible promoter that has been designed for high expression in ''B.subtilis''. Gene expression under the promoter hyper-spank can be induced by addition of Isopropyl β-D-1-thiogalactopyranoside (IPTG). The context with which we used the promoter hyper-spank, was to take an input of IPTG and give '''Polymerase Per Second'''(PoPS) as an output. IPTG does not induce the promoter hyper-spank directly, but requires the transcriptional regulator '''LacI''', (<bbpart>BBa_K413035</bbpart>). This means that LacI must be constitutively expressed in ''B.subtilis'' in order to use the promoter hyper-spank.
Biobrick standard was applied to the promoter hyper-spank sequence.
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.
Conversion of the iGEM parts registry to SBOL2.1