Sensory rhodopsin II (SRII) with HtrII & Tsr, sensitive to blue-green light
Sensory rhodopsin II fusion with HtrII & Tsr, which is sensitive to blue-green light, with sensing spectra covering from 400-500 nm. We used it for sensing blur-green light to achieve phototaxis and switch for downstream gene expression.
Pfam (version 26.0) was used to predict the domain of fusion protein. Linker was refereed and modified from previous research study of SRII fusion with HtrII.
From genomic sequence of bacterial N. pharaonis (DSM 2160) and E.coli K12.
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
with a Ptet promoter controled by TetR, a MCS, T1 terminator, p15A ori and Cm resistence gene. copy number: 15~20
to measure the efficiency of different SD signal, the plasmid has a MCS downstream of Ptet where can insert reporters along with SD signal. There is a T1 terminator downstream of MCS to terminate transcription starting from Ptet. Ptet is repressed by TetR carried on this plasmid and can be turned on by adding aTc.
T7 promoter-driven BMC structural genes with a His6-tagged component for purification
T7 promoter diving the expression of His6-tagged EutS, with subsequent Plac-driven EutM, EutN, EutL, EutK
Clone BBa_K311004 into pET28a via NheI and NdeI
The part consists of a sequences that resemble a T7 promoter and the lacO sequence found in E. coli.
This is a mutant T7 promoter that includes a lacO sequence. This is repressible by the LacI protein and is induced in the presence of IPTG. The promoter sits in the 5' region of the gene of interest and initiates transcription when the cell expresses T7 RNA polymerase. Be sure you are using a cell strain that includes this gene (DE3).
There are three portions to the T7 promoter: the recognition site, the melting box (or TATA box) and the initation site. Mutations in these sequences caused differential behavior from the wild type.
Halorhodopsin come from N.pharaonis, gene bank J05199.1
Halorhodopsin under constitute T7 promoter to regulate the gene transcription to be maximized and no restrict to other regulatory factor
The coding sequence of N.pharaonis J05199.1 is selected from 61nt to 700nt and the T7 promotor placed in front by 3A assembly
OmpA is derived from the genomic DNA of BL21 (DE3) E. Coli strain while SH3 is from human Grb2.
This part contains the N-terminal 180 amino acids of OmpA fused to SH3 domain in human Grb2 protein. The N-terminal of OmpA allows the moiety fused to it to be presented on to the outer membrane of E. Coli. SH3 domain allows for binding to multi-proline peptide in human cells, which functions as a crucial adaptor module in signaling. In our part, this is specially for binding to the proteins for transportation by E. Coli. All the coding sequence is under T7 promoter and lac repressor binding site. The parts is most efficiently presented onto the membrane when induced at 0.1mM IPTG at 18 centigrade in BL21 E. Coli strain.
High expression efficiency as well as inducible expression is most desirable when considering a coding sequence.
beta-lactamase (ampicillin resistance marker)
HIV protease sequence is from former part BBa_I712667. OmpA sequence is from BL21(DE3) e. Coli strain.
OmpA is used to present HIV aspartyl protease outside the E. Coli membrane so that it can cut other proteins with the specific sequence outside in the media.
Since HIV protease is a dimer, dimerization is a great concern to its function. Function of this protease is tested in BL21.
Sensory rhodopsin II (SRII) with HtrII & Tar, sensitive to blue-green light
Sensory rhodopsin II fusion with HtrII & Tar, which is sensitive to blue-green light, with sensing spectra covering from 400-500 nm. We used it for sensing blur-green light to achieve phototaxis and switch for downstream gene expression.
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.
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.
T7 terminator for protein expression in E. coli or in vitro. Part is provided in R.F.C. 25 assembly format.
* taken from pET3a expression plasmid * added two STOP to 5' * conservative choice of part boundary, could probably be shortened
OmpA is from BL21(DE3) E. Coli genomic DNA. SH3 is from human Grb2 protein. HIV protease site is from a former part.
Added HIV protease site between OmpA and SH3 for easier release.
The HIV protease site is embedded in the protein so that a proteolytic cleavage can release the binding cassette.
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
T7 promoter for expression of functional RNA
Red light triggering phototaxis using chp8
Red light triggering phototaxis using chp8, which can sense the red light from 600-700 nm.
6 poly-His tag was positioned at C-terminal of the protein.
The sequence of chp8 is from previous biobrick.
luxABCDE gene ligated to the rear of T7 promoter. When transformed into BL21(DE3) E.coli strain, add different amount IPTG to reach different final concentration of IPTG in the medium can induce different level of Lux ligt emission. No substrate is needed.
luxABCDE gene ligated to the rear of T7 promoter. The original Ara and P-bad were replaced with T7 promoter, which enables its usage in our project.
T7 promoter and LuxCDABE with their RBS are from Partsregistry.
LuxCDABEG with corresponding six RBS(from K325909)
Inducible High Expression His-Tagged Photosystem II CP47 subunit
Halorhodopsin comes from part BBa_K559000, which is come from genomic sequence of N.pharaonis (genebank ID:J05199.1)
Halorhodopsin gene allow chloride ion to be pumped in by the level of light intensity. Halorhodopsin gene is under regulation by constitute T7 promoter to allow protein coding to be maximized, and the bidirectional terminator is added for high efficiency termination of both forward and backward transcription
The double terminator wass tested to stop effectively on the transcription of both forward and backward transcription to make our protein generator be a controlled system
This is our expression vector. The plasmid has the restriction sites XbaI NgoMIV_part_AgeI_SpeI_NotI_PstI
By adding additional restriction sites in pMAL we create our expression vector.
Sensory rhodopsin I (SRI) with HtrII & Tar, sensitive to orange light
Sensory rhodopsin I fusion with HtrII & Tar, which is sensitive to orange light, with sensing spectra covering from 590-650 nm. We used it for sensing blur-green light to achieve phototaxis and switch for downstream gene expression.
Pfam (version 26.0) was used to predict the domain of fusion protein. Linker was refereed and modified from previous research study of SRI fusion with HtrII.
Strong T7 promoter and RBS including start codon for protein expression in E. coli or in-vitro. Part is provided in RFC 25 assembly format.
* sequence is taken from the commonly used pET3a expression vector * deleted XbaI site between prom. and RBS * T7 promoter sequence (1-19) equals the one in BBa_I712074 * Conservative choice of part boundaries and modifications -- the part's size could probably be reduced.
The 'repeat' sequence in CRISPR/cas system of E.coli strain K12.
It is a repeat-spacer-repeat region designed according to E.coli's CRISPR/Cas system. It also includes a T7 promoter and T7 terminator. THe spacer is designed to target on a segment of non-coding-sequence in E.coli's genome. This biobrick can be transformed into E.coli cells to hopefully construct a suicide system with E.coli's CRISPR/Cas system.
The repeat-spacer-repeat region design should be consistent with the operating principles of E.coli's CRISPR/cas system.
Double terminator consisting of BBa_B0012 and BBa_B0011
Derived from luxICDABEG operon terminator of Vibrio fischeri <genbank>AF170104</genbank>.
Bidirectional transcriptional terminator consisting of a 22 bp stem-loop.
In the naturally-occuring sequence there is a mismatch in the stem of the stem loop. This can be corrected via an A->G mutation (at position 40 -- sequence coordinate/not MFOLD coordinate). The above sequence does not reflect this mutation (but the MFOLD image does). This terminator's location cannot be found using some inverted repeat detectors like PALINDROME because it is too short and contains a mismatch. This one was found with the help of Tom Knight. It lies between two coding regions that point towards eachother.
CP47 His-tagged with Lac Promoter synthesized by IDT DNA Inc.
Conversion of the iGEM parts registry to SBOL2.1