Anderson promoter J23118 without RFP in backbone vector pSB1C3 to easily fuse the promoter with other reporters e.g. the lux operon.
PcotYZ: B. subtilis promoter regulating expression of spore crust proteins
PcotYZ: B. subtilis promoter regulates spore crust proteins and is activated in late sporulation. PcotYZ regulates the crust protein cotZ and cotY.
We use this promoter for the expression of fusion proteins on B. subtilis spores.
Anderson promoter J23117 without RFP in backbone vector pSB1C3 to easily fuse the promoter with other reporters e.g. the lux operon.
Anderson promoter J23102 without RFP in backbone vector pSB1C3 to easily fuse the promoter with other reporters e.g. the lux operon.
Anderson promoter J23113 without RFP in backbone vector pSB1C3 to easily fuse the promoter with other reporters e.g. the lux operon.
pSBBs0K-Pspac (replicative Bacillus subtilis expression vector; IPTG inducible
This part is an expression vector for Bacillus subtilis. It is replicative both in E.coli and B.subtilis. It has a ampicillin resistance for cloning in E.coli and kanamycin resistance for selection in B. subtilis. The multiple cloning site is downstream of a Pspac promoter, that is inducible with IPTG. (Isopropyl-β-D-thiogalactopyranosid). This BioBrick is part of the LMU 2012 igem project Bacillus BioBrickBOX http://2012.igem.org/Team:LMU-Munich/Bacillus_BioBricks
We removed any forbidden restriction sites by site-directed mutagenesis PCR. The clonging site also includes a rfp for selection for cloning in E.coli. It can be used as an inducible expression vector in B. subtilis.
This part is an igem compatible version of the Bacillus expression vector pDG148
Lambda Beta recombinase optimized for expression in B. subtilis
Recombination-mediated genetic engineering (recombineering) utlises homologous recombination to facilitate genetic modifications at any desired target by flanking the mutated sequence with homologous regions. Multiplex Automated Genome Engineering (MAGE) is a method for rapid and efficient targeted programming and evolution of cells through cyclical recombineering using multiple single-stranded DNA oligonucleotides (oligos). The MAGE protocol utilises the λ Red recombination system in combination with an (temporary) inactivation of the mismatch repair system and consists of 7 steps that can be done with standard laboratory equipment (Wang, 2009). As MAGE utilises oligos, only the Beta protein of the λ Red system is required. This BioBrick encodes an optimised coding sequence for lambda phage derived beta recombinase. When expressed in Bacillus subtilis, oligo recombineering efficiencies were increased. (Please see results).
The original nucleotide sequence from E. coli () was optimized for expression in Bacillus subtilis using JCat
Anderson promoter J23106 without RFP in backbone vector pSB1C3 to easily fuse the promoter with other reporters e.g. the lux operon.
2-oxoglutarate oxygenase/decarboxylase is an Ethylene Forming Enzyme (EFE) that catalyze Ethylene biosynthesis from 2-oxoglutarate. This part is in the pSpac vector that has benn sent to the Igem UNITN 2013 team from the LMU Munich 2012 team. This part was cloned by the iGEM Trento 2013 team for the creation of an aerobically engineered pathway for the control of fruit ripening. Further information about this part and its characterization can be found in the iGEM Trento 2013 wiki.
The coding sequence was taken from Pseudomonas syringae pv. phaseolicola PK2. We firstly optimized the codons for both B. subtilis and E. coli and then synthesized the gene with an RBS sequence upstream of the start codon. We decided to include in the CDS two restriction sites (NgoMIV and AgeI) in order to allow people to use the Freiburg assembly.
This enzyme was firstly purified from Pseudomonas Siringae pv. phaseolicola PK2, a 2-oxoglutarate-dependent ethylene producing bacterium [1]. The CDS has been optimized for both E.coli and B.subtilis. We decided to include an RBS sequence too.
Anderson promoter J23114 without RFP in backbone vector pSB1C3 to easily fuse the promoter with other reporters e.g. the lux operon.
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.
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.
Anderson promoter J23100 without RFP in backbone vector pSB1C3 to easily fuse the promoter with other reporters e.g. the lux operon.
Anderson promoter J23101 without RFP in backbone vector pSB1C3 to easily fuse the promoter with other reporters e.g. the lux operon.
Gram-Positive Strong Constitutive Promoter
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.
We PCRed this part out of the pAD43-25 gram positive vector, acquired from the Bacillus Genetic Stock Center (bgsc.org), catalog number ECE166.
Promoter gsiB is a sigma factor B dependent promoter found in ''B.subtilis''. In ''B.subtilis'' endogenous sigma factor B is activated under mild stress. These mild stress conditions can be generally split into nutrient stress response and physical stress response. Nutrient stress response is triggered by low levels of ATP and GTP and physical stress response is triggered by exposure to blue light, salt, heat, acid or ethanol1. The promoter gsiB has been used previously as a read out for the activation of sigma factor B 2. The context with which we used the promoter gsiB, was to take blue light as an input and give '''Polymerase Per Second'''(PoPS) as an output. To do this the other potential inputs need to be carefully controlled so that only blue light activated the sigma B and gives a PoPS output. In order to get sufficient sigma B activation by blue light the light receptor YtvA, part...., needs to be over expressed in ''B.subtilis'' 3.
Biobrick standard was applied to the promoter gsiB sequence.
The part was designed using the sequence from the ''B.subtilis'' genome and from previously published papers 23. 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.
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.
Anderson promoter J23103 without RFP in backbone vector pSB1C3 to easily fuse the promoter with other reporters e.g. the lux operon.
This is an IPTG-inducible 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.
We found the functional annotation in the associated paper: Kaltwasser M., T. Wiegert, and W. Schumann. 2002. Construction and Application of Epitope- and Green Fluorescent Protein-Tagging Integration Vectors for Bacillus subtilis. Appl. Environ. Microbiol 68:2624-2628 The primers we used to extract it (sans biobrick ends) are as follow: Forward: AGAACAACCTCTGCTAAAATTC Reverse: AAGCTTAATTGTTATCCGCTC
We PCRed this part out of the pMUTIN-YFP gram positive vector, acquired from the Bacillus Genetic Stock Center (bgsc.org), catalog number ECE151.
Promoter ctc is a sigma factor B dependent promoter found in ''B.subtilis''. In ''B.subtilis'' endogenous sigma factor B is activated under mild stress. These mild stress conditions can be generally split into nutrient stress response and physical stress response. Nutrient stress response is triggered by low levels of ATP and GTP and physical stress response is triggered by exposure to blue light, salt, heat, acid or ethanol1. The promoter ctc has been used previously as a read out for the activation of sigma factor B 2. *The context with which we used the promoter ctc, was to take blue light as an input and give '''Polymerase Per Second'''(PoPS) as an output. To do this the other potential inputs need to be carefully controlled so that only blue light activated the sigma B and gives a PoPS output. In order to get sufficient sigma B activation by blue light the light receptor YtvA, part...., needs to be over expressed in ''B.subtilis'' 3. ===Reference=== <biblio> #1 pmid=16267279 #2 pmid=3100810 #3 pmid=17575448 </biblio>
Biobrick standard was applied to the promoter ctc sequence.
Promoter 43 a constitutive promoter for B. subtilis
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.
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.