pSBBs1C: Empty backbone for integration into Bacillus subtilis amyE locus
This part is an empty backbone vector for the usage in Bacillus subtilis. It integrates in the amyE locus and can be selected with Chloramphenicol (cat gene). It has a ampicillin resistance for cloning in E.coli. It originates from the Bacillus vector pDG1662. This BioBrick is part of the LMU 2012 Bacillus BioBrick Box project: http://2012.igem.org/Team:LMU-Munich/Bacillus_BioBricks
We removed any forbidden restriction sites by site-directed mutagenesis PCR.
This backbone is an igem compatible version of the Bacillus vector pDG1662
BioScaffold Linker - Removes Stop Codons & scars & replaces with a Gly-Ser linker
This part is part of a family of Bioscaffold - Linkers. This part will help users interested in protein-fusion. The usage of the part will allow in frame assembly of 2(+) proteins by: *Removing the stop codons from the upstream protein. *Removing the scar created by the Assembly Standard 10 (RFC10) construction of 2(+) protein coding sequences. *Maintaining the start codon of the downstream protein. *Adding a flexible (Gly-Ser-Gly) linker between the two protein coding sequences. *Acting as a translational buffer if ligation is incorrect. ===Assembly Instructions=== *Create an Assembly Standard 10 fusion as below ; **Upstream Protein Part - Bioscaffold Linker - Downstream Protein Part** *Use the BioScaffold Specific Enzymes (i)Restrict with BpuEI to remove upstream scar (created between Upstream Protein - Bioscaffold) and stop codons from upstream protein part. (ii)Ligate and you will get the intermediate product; **Upstream Protein (w/out Stop codons) - Tyr - BioScaffold Linker - Downstream Protein** *Restrict with CspCI to remove downstream scar. *Ligate to get the final product; **Upstream Protein - Tyr-Gly-Ser-Gly- Downstream Protein** ====N.B.==== CspCI cuts with a 1 b.p discrepancy depending on the sequence. However this should not affect the restriction/ligation process as this has been taken into account and incorporated into the design. Even with the discrepancy involved the restriction/ligation process will still give the same product and in-frame fusion. The other restriction variants of CspCI have also been taken into account and relevant BioScaffolds are being produced to be tested. Updates will soon follow on this. This version accounts for cleaving DNA 12/10bp (top/bottom) on the 5' of the recognition sequence and 10/12bp (top/bottom) on the 3' site.
CspCI enzyme has different variations in the distance of restricting DNA from the recognition site.This biobrick is almost identical to part BBa_K259004 but modified to account for this discrepancy.
This part is completely designed de novo. This sequence does not occur naturally.
FRET based ATP biosensor for expression in Aspergillus niger
Consists of cfp-yfp fret pair that responds to changes in ATP concentration
It has illegal restriction sites for the standard 10 assembly. The cds of the part is flanked by NdeI and NotI restriction sites.
Integration vector for Bacillus subtilis derived from pSac-Cm
The plasmid is designed to integrate a cloned insert into the B. subtilis 168 chromosome at the sacA locus. The user inserts the fragment inside the MCS whi
This part is part of a family of Bioscaffold - Linkers. This part will help users interested in protein-fusion. The usage of the part will allow in frame assembly of 2(+) proteins by: *Removing the stop codons from the upstream protein. *Removing the scar created by the Assembly Standard 10 (RFC10) construction of 2(+) protein coding sequences. *Maintaining the start codon of the downstream protein. *Adding a flexible (Gly-Ser-Gly) linker between the two protein coding sequences. *Acting as a translational buffer if ligation is incorrect. ===Assembly Instructions==== *Create an Assembly Standard 10 fusion giving ; **Upstream Protein Part - Bioscaffold Linker - Downstream Protein Part *Use the BioScaffold Specific Enzymes **Restrict with BpuEI to remove upstream scar (created between Upstream Protein - Bioscaffold) and stop codons from upstream protein part. **Ligate and you will get the intermediate product; Upstream Protein (w/out Stop codons) - Tyr - BioScaffold Linker - Downstream Protein **Restrict with BseRI to remove downstream scar. **Ligate to get the final product; Upstream Protein - Tyr-Gly-Ser-Gly- Downstream Protein
Part was designed taking into account RFC10 assembly standard and as an attempt to be upwards compatible with RFC10.
This part was designed ''de novo''. The sequence does not occur naturally.
Tyr Residue involved in Restriction/Ligation
pSBBs2E: Empty backbone for integration into Bacillus subtilis lacA locus
This part is an empty backbone vector for the usage in Bacillus subtilis. It integrates in the lacA locus and can be selected with erythromycin + lincomycin (MLS). It has a ampicillin resistance for cloning in E.coli. This BioBrick is part of the LMU 2012 Bacillus BioBrick Box project: 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 in E.coli.
This part is an igem compatible version of the Bacillus subtilis vector pAX01
composite part of Bba_K365005 and Bba_K365000
pBS2E: Bacillus subtilis vector, lacA integration, MLS resistance
This part is an empty backbone vector for the usage in Bacillus subtilis. It integrates in the lacA locus and can be selected with erythromycin + lincomycin (MLS). It has a ampicillin resistance for cloning in E.coli.
Compatible with pBs1C and pBs4S in one strain.
This backbone is a BioBricked version of the B. subtilis vector pAX01. This BioBrick is part of the LMU 2012 igem project [http://2012.igem.org/Team:LMU-Munich/Bacillus_BioBricks '''''Bacillus''B'''io'''B'''rick'''B'''ox] Reference: [http://www.ncbi.nlm.nih.gov/pubmed/11274134 H??rtl ''et al.'']
pSBBs1C-lacZ (lacZ reporter vector for B. subtilis)
This part is a backbone vector for Bacillus subtilis. It integrates at the amyE locus. It has a Ampicillin resistance for cloning in E.coli and Chloramphenicol resistance for B.subtilis. The multiple cloning site is followed by a lacZ gene for reporter assays. This BioBrick is part of the LMU 2012 igem project Bacillus BioBrick Box http://2012.igem.org/Team:LMU-Munich/Bacillus_BioBricks
This reporter vector can be used with the normal BioBrick assembly strategies.
This vector is the Bacillus vector pAC6 brought into the iGEM BioBrick standard.
pBS1C: Bacillus subtilis vector, amyE integration, CAM-resistance
This vector integrates into the amyE-site of Bacillus subtilis via double recombination. The two recombination sites flank a standard BioBrick multiple cloning site (with RFP-cassette), as well as a chloramphenicol resistance cassette (5 ??g/ml). The backbone does not contain the restriction sites EcoRI, XbaI, SpeI, PstI, NgoMIV nor AgeI. The vector is replicative in Escherichia coli and provides resistance to ampicillin (100 ??g/ml). Before transformation into B. subtilis, this vector should be linearized with ScaI.
This is one of three empty vectors with compatible integration loci and resistance cassettes to allow combination of up to three constructs of choice in one single strain.
This part is part of a family of Bioscaffold - Linkers. This part will help users interested in protein-fusion. The usage of the part will allow in frame assembly of 2(+) proteins by: *Removing the stop codons from the upstream protein. *Removing the scar created by the Assembly Standard 10 (RFC10) construction of 2(+) protein coding sequences. *Maintaining the start codon of the downstream protein. *Adding a flexible (Gly-Ser-Gly) linker between the two protein coding sequences. *Acting as a translational buffer if ligation is incorrect. ===Assembly Instructions=== *Create an Assembly Standard 10 fusion as below ; **Upstream Protein Part - Bioscaffold Linker - Downstream Protein Part** *Use the BioScaffold Specific Enzymes (i)Restrict with BpuEI to remove upstream scar (created between Upstream Protein - Bioscaffold) and stop codons from upstream protein part. (ii)Ligate and you will get the intermediate product; **Upstream Protein (w/out Stop codons) - Tyr - BioScaffold Linker - Downstream Protein** *Restrict with CspCI to remove downstream scar. *Ligate to get the final product; **Upstream Protein - Tyr-Gly-Ser-Gly- Downstream Protein**
This part was designed taking into consideration the requirements by Assembly Standard 10 (RFC10).
This part is completely designed ''de novo''. This sequence does not occur naturally.
pSBBs3C-luxABCDE (lux reporter vector for B.subtilis)
This part is a reporter vector for Bacillus subtilis. It integrates at the sacA locus of B.subtilis genome. It has a ampicillin resistance for cloning in E.coli and Chloramphenicol resistance for selection in B. subtilis. The multiple cloning site has a rfp for selection and is followed by the luxABCDE gene cluster for luminescence reporter assays. This BioBrick is part of the LMU-Munich 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.
This vector is an igem compatible version of the pAH328 Bacillus reporter vecotr.
2-oxoglutarate oxygenase/decarboxylase is an Ethylene Forming Enzyme (EFE) that catalyze Ethylene biosynthesis from 2-oxoglutarate. This part is in the pXyl vector that has been 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.
===Design Notes=== 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. GAATTCGCGGCCGCTTCTAGATAAGGAGGAACTACTATGGCCGGCACC... ...TCGACCGGTTAATACTAGTAGCGGCCGCTGCAG
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.
Plasmids containing oriV and the corresponding Rep proteins (from RSF1010) are autonomously replicated in a broad range of hosts. It has been reported that RSF1010 derived plasmids containing oriV and the Rep proteins are stably maintained in Pseudomonas (Bagdasarian 1981), Caulobacter (Umelo-Njaka et al. 2001), Erwinia, Serratia (Leemans 1987), and several cyanobacteria strains (Mermet-Bouvier 1993) including Synechocystis PCC6803 and PCC6714 and Synechococcus PCC7942 and PCC6301.
This part is to be constructed with the Rep proteins derived from an RSF1010 plasmid.
This part is taken from the RSF1010 derived plasmid pRL1383a. A description of this plasmid can be found at the NCBI database at accession number AF403426.
Multi-host vector pTG262 converted to BioBrick vector
biobricked version of pTG262 Plasmid has a multi host replication origin and is competant in range of gram positive and gram negative bacteria.
We kindly thank Dr Mike Gasson & Dr. Claire Shearman, of the Institute of Food Research in Norwich for generously donating the pTG262 vector.
This part is part of a family of Bioscaffold - Linkers. This part will help users interested in protein-fusion. The usage of the part will allow in frame assembly of 2(+) proteins by: *Removing the stop codons from the upstream protein. *Removing the scar created by the Assembly Standard 10 (RFC10) construction of 2(+) protein coding sequences. *Maintaining the start codon of the downstream protein. *Adding a flexible (Gly-Ser-Gly) linker between the two protein coding sequences. *Acting as a translational buffer if ligation is incorrect. ===Assembly Instructions=== *Create an Assembly Standard 10 fusion as below ; **Upstream Protein Part - Bioscaffold Linker - Downstream Protein Part** *Use the BioScaffold Specific Enzymes (i)Restrict with BpuEI to remove upstream scar (created between Upstream Protein - Bioscaffold) and stop codons from upstream protein part. (ii)Ligate and you will get the intermediate product; **Upstream Protein (w/out Stop codons) - Tyr - BioScaffold Linker - Downstream Protein** *Restrict with CspCI to remove downstream scar. *Ligate to get the final product; **Upstream Protein - Tyr-Gly-Ser-Gly- Downstream Protein** ====N.B.==== CspCI cuts with a 1 b.p discrepancy depending on the sequence. However this should not affect the restriction/ligation process as this has been taken into account and incorporated into the design. Even with the discrepancy involved the restriction/ligation process will still give the same product and in-frame fusion. The other restriction variants of CspCI have also been taken into account and relevant BioScaffolds are being produced to be tested. This version is for when CspCI cuts 13/10b.p. (top/bottom) on the 5' of the recognition sequence and 10/13bp (top/bottom) on the 3' of the recognition sequence.
The CspCI discrepancy had to be taken into account
Broad host range BioBrick plasmid derived from pRL1383a with low to medium copy number
RSF1010 is a broad-host-range plasmid first described in 1974 (Guerry 1974), with its entire sequence and gene organization subsequently described in 1989 (Scholz 1989). It is a naturally occurring 8.6kb broad-host-range plasmid in the E. coli incompatibility group Q. The conjugative transfer and stable replication of this plasmid are possible due to the mob genes with the associated origin of transfer (oriT) and the rep genes with the associated origin of vegetative replication (oriV), respectively. RSF1010 derived plasmids which include the oriV and associated Rep proteins are stably maintained in Pseudomonas (Bagdasarian 1981), Caulobacter (Umelo-Njaka et al. 2001), Erwinia, and Serratia (Leemans 1987). In addition, RSF1010 derived plasmids including the oriV, oriT and its associated rep and mob genes are transferred by conjugation to at least four cyanobacteria strains (Mermet-Bouvier 1993). These cyanobacteria strains include Synechocystis PCC6803 and PCC6714 and Synechococcus PCC7942 and PCC6301. A number of RSF1010 derived plasmids have been constructed due to the utility of this broad-host-range plasmid. For example, pSB2A, containing a 5.6kb RSF1010 derived region including the necessary mobilization and replication regions, can be transferred through conjugation to and stably maintained in Synechocystis PCC6803, PCC6714, and Synechococcus PCC7942 and PCC6301 (Marraccini 1993). The RSF1010 plasmid used in this study, pRL1383a was constructed for use in genomic studies of the diazotrophic, multicellular cyanobacterium Anabaena PCC 7120. This plasmid contains the mob and rep regions necessary for conjugation and autonomous replication, respectively. Additionally this vector is made resistant to Streptomycin and Spectinomycin due to the presence of the aadA gene (Wolk 2007). This plasmid can be mobilized by the E. coli derived self-transmissible plasmid RP4. Mobilization genes are not necessary for transfer of a mobilizable plasmid if the self-transmissible plasmid and the mobilizable plasmid share a common origin of transfer (Snyder and Champness 2007). The origin of transfer for RP4 is 99 base pairs and contains binding sites for transfer proteins encoded by RP4. For more information and a list of references, please visit: http://2008.igem.org/Team:Hawaii/Project/Part_A#Literature_Cited
The original pRL1383a MCS has been replaced by the BioBrick BBa_B0034 and its flanking BioBrick restriction sites.
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.
Phytochrome B (PhyB, first 908 N-terminal amino acids)
improved version of BBa_K365002 to allow N- and C-terminal protein fusions using RFC 25
Essential Arg for photoconvertible binding of PIF3/6
Essential Gly for photoconvertible binding of PIF3/6
Essential Cys for chromophore attachement of phycocyanobilin/phytochromobilin
pSBBs4S-Pxyl: Integrative expression vector for Bacillus subtilis</i
This part is an expression vector for Bacillus subtilis. It integrates at the thrC locus. It has a ampicillin resistance for cloning in E.coli and chloramphenicol resistance for selection in B.subtilis. The multiple cloning site downstream of a Pxyl promoter, that is inducible with xylose. This BioBrick is part of the LMU 2012 igem project Bacillus BioBrick Box http://2012.igem.org/Team:LMU-Munich/Bacillus_BioBricks
This part is an igem compatible version of the Bacillus expression vector pXT
FRET based ATP biosensor cds, codon optimized for Aspergillus niger
Shreyans Chordia (shreyanschordia90@gmail.com)
A FRET based biosensor that will measure the ratio of ATP and ADP.
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
pBs4S: Bacillus subtilis vector, thrC integration, spec resistance
This part is an empty backbone vector for the usage in Bacillus subtilis. It integrates in the thrC locus and can be selected with Spectinomycin cassette. It has a ampicillin resistance for cloning in E.coli. This backbone is a BioBricked version of the B. subtilis vector pDG1731.
This vector is compatible with pBS1C and pBS2E in one strain.
This BioBrick is part of the LMU 2012 igem project BacillusBioBrickBox For handling of B. subtilis vectors, please see here. The transformation into B. subtilis is explained here.
pSBBs4S: Empty backbone for integration into Bacillus subtilis thrC locus
This part is an empty backbone vector for the usage in Bacillus subtilis. It integrates in the thrC locus and can be selected with Spectinomycin cassette. It has a ampicillin resistance for cloning in E.coli. It originates from the Bacillus vector pDG1731.
This plasmid can be used as empty backbone vectors like pSB1C3 but is able to integrate in B.subtilis. The clonging site also includes a rfp for selection.
composite part of Bba_K365000 and Bba_K365005
This plasmid was derived from pSac-Cm by insertion of biobrick compatible restriction sites (prefixes and suffixes), a terminator (BBa_B0015) after the suffixes sequences, and red fluorescent protein sequence (RFP) in between the prefix and suffix in its multiple cloning sites (MCS). New biobricks can be inserted into this vector by replacement of the RFP biobrick, and selection of the white colonies. This backbone has a multi host replication origin and replicates in E. coli. The plasmid is designed to integrate a cloned insert into the B. subtilis chromosome via double recombination between plasmid and chromosomal sacA sequences, and in order to check for integration a simple Phenol Red Sucrose test can be perform, if sacA is no longer functional, they will remain ph7, if they are still able to digest sucrose their pH will be under 5.
We found that that the integration plasmid that groningen send to the registry were unable to integrate in B. subtilis. This is due to the high number of mutations in the sacA integration region.