pSB4A50: Low copy BioBrick cloning/expression backbone carrying Amp resistance
Constantin Ahlmann-Eltze, Charlotte Bunne, Magdalena B??scher, Jan Gleixner, Max Horn, Anna Huhn, Nils Klughammer, Jakob Kreft, Elisabeth Sch??fer, Carolin Schmelas, Silvan Schmitz, Max Waldha
The part BBa_J04500 was used as a template for the creation of this new part. The -35 and -10 promoter elements were converted through mutagenesis into AflII and ClaI (respectively) restriction sites. A double restriction digest was performed and a T7 promoter was introduced in the form of primers.
We wanted a T7 promoter that could be regulated with the lac system.
The part BBa_J04500 was used as a template for the creation of this new part
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
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 part is an igem compatible version of the Bacillus expression vector pXT
pSB4C50: Low(?) copy BioBrick cloning/expression backbone carrying Cm resistance
R0011+B0034, strong IPTG-inducible promoter with strong RBS.
This part contains the strong, IPTG-inducible promoter R0011, combined with the strong RBS B0034. This part can be used as a strong protein expression system when combined with a protein coding sequence.
This part should offer greater levels of expression when compared to similar parts using the natural lac promoter (R0010).
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.
3OC6HSL+LuxR dependent POPS/RIPS generator
produces pops and rips in the presence of luxR/3OC6HSL and the absence of lambda cI.
We take advances in genetic engineering as synthetic biology, as viable solution to oil pollution cleanup. But for do this; we need to design and build an expression platform that works into our modified E.coli. For this, We choose four standardized biological parts from the Registry, those parts are: IPTG inducible promoter, Ribosomal Binding Site (RBS), GFP Reporter and Double Terminator; Were selected using rationale from molecular biology and genetic engineering to enable us to build up rationally-engineered, our expression platform. For the iGEM 2011, we will integrate and assembly those parts (P+RBS+GFP+T) along with our BioBrick part using the 3A Assembly Method, to bring up an expression device (P+RBS+rhlAB_BB+GFP+T) for rhamnolipid production in E. coli.
Those parts were assembled following the 3A assembly method.
IPTG inducible promoter with RBS http://partsregistry.org/wiki/index.php?title=Part:BBa_J04500 GFP reporter plus Double Terminator http://partsregistry.org/wiki/index.php/Part:BBa_K653001
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
pSB1A30: High copy BioBrick cloning/expression backbone carrying Amp resitance
Expression Plasmid coding for His-tag, also containing a thrombine cleavage site between coding region and Histag, so that His-tag can be cleaved off. Vecotr opening with PstI/XmaI, clone part into it with PstI/NgoMIV. Ampicillin resistance.
very strong RBS 6xHis-Tag Thromin-cleavage-site NgoMIV-compatible restriction site for in-frame insertion
pTUM104 yeast shuttle vector with GAL1 promoter
This part is based on the plasmid pTUM100 (BBa_K801000). The galactose-inducible GAL1 yeast promoter has been cloned in front of the MCS (RFC25 prefix and suffix).
All forbidden restriction sites have been removed, making this plasmid RFC25-compatible.
Based on invitrogen's pYES2 plasmid. For more information see the part information of pTUM100 (BBa_K801000).
Plambda is a repressible promoter, cI regulated (BBa_C0051). Without cI, the promoter is ON with strong activity. RBS is strong (efficiency=0.6)
Plambda: Vinay S Mahajan, Brian Chow, Peter Carr, Voichita Marinescu and Alexander D. Wissner-Gross RBS:
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
Low copy BioBrick expression backbone carrying Kan resistance
Constantin Ahlmann-Eltze, Charlotte Bunne, Magdalena Büscher, Jan Gleixner, Max Horn, Anna Huhn, Nils Klughammer, Jakob Kreft, Elisabeth Schäfer, Carolin Schmelas, Silvan Schmitz, Max Waldha