meso-2,6-Diaminopimelic acid (mDAP) is a non-proteinogenic amino acid that is synthesized and se-creted by Chlamydia trachomatis. mDAP has a relatively high chemical similarity to aspartate, sharing a Tanimoto coefficient of T=0.8 in a molecular fingerprint analysis [T<0: chemically very different struc-tures; T=1: identity]. As a proof of concept, a molecular docking model proposes few mutations which would change the Tar specificity towards mDAP. As input for molecular docking we used the crys-tal structure of the ligand binding domain of Tar bound to aspartate (PDB: 4Z9H). As a base for the part sequence, the BioBrick representation of the original Taz, BBa_C0082, was used. The BBa_C0082 BioBrick features a small part of the vector (64 nucleotides) it was derived from on its 3??? end, which we have removed along with the stop codon inside the Part for further fusion protein de-sign in RFC 12, 21, 23 and 25 assembly standards. We used the Glide algorithm, Maestro and the Schrodinger Suite for molecular docking of aspartate and mDAP to the Tar ligand binding domain (PDB: 4Z9H). Because of its structural similarity to aspartate, mDAP showed some affinity to the native Tar aspartate binding site (Table 1). Since mDAP is larger than aspartate, Y149 led to structural interference, not allowing mDAP to fully enter the binding site. To create the additional space needed for mDAP binding, we substituted Y149 with smaller amino acids. The best results were obtained with serine. Because of the additional size of mDAP, the bigger challenge was to reduce the affinity of aspartate rather than to raise the affinity of mDAP. We identified R64 as a key residue for aspartate binding which hovewer was not needed to bind mDAP. To minimally interfere with folding and function we performed a constitutive substitution and substituted it with lysine, creating the R64K Y149S double mutant called mTaz hereafter. Using mTaz, a higher docking score was achieved for mDAP than for aspartate (Table 1).
mTaz is based on the Tar-EnvZ chimeric receptor which is capable of detecting aspartate in the medium as described by Utsumi et al. 1989. Tar, or methyl-accepting chemotaxis protein II, is a signal transducer in Escherichia coli chemotaxis and is activated upon the recognition of aspartate. Tar is sensitive to mutations altering its ligand specificity which sometimes results in recognizing other amino acids. EnvZ is a part of the well-studied and commonly used two-component system EnvZ/OmpR in E. coli, offering a simple way of signal transduction. Its periplasmic domain was shown to be exchangeable with different sensor domains e.g. Tar/EnvZ chimeric receptor capable of detecting aspartate in the medium.
TNT-R3: a computationally designed receptor binding TNT, made by mutation of the Escherichia coli periplasmic ribose-binding protein: see Looger, L., Dwyer, M.A., Smit, J.J., and Wellinga, H.W. 2003. Computational design of receptor proteins with novel functions. Nature 423, 185-190. This receptor interacts with a hybrid transmembrane protein Trz, created by combining the normal partner of ribose-binding-protein, Trg, with the intracellular part of 2-component sensor protein EnvZ, which normally phosphorylates response regulator protein OmpR, allowing it to activate promoters such as PompC (see Baumgartner, J.W., Kim, C., Brisette, R.E., Inouye, M., Park, C., and Hazelbauer, G.L. 1994. Transmembrane signalling by a hybrid protein: communication from the domain of chemoreceptor Trg thate recognizes sugar-binding proteins to the kinase/phosphatase domainof Osmosensor EnvZ. J. Bacteriol. 176, 1157-1163). To use this receptor, include it in a system with Trz and a reporter gene controlled by PompC.To avoid cross-talk, it may be necessary to use a chassis lacking EnvZ and ribose-binding protein.
Derived by mutagenesis of the Escherichia coli periplasmic ribose-binding protein: see Looger, L., Dwyer, M.A., Smit, J.J., and Wellinga, H.W. 2003. Computational design of receptor proteins with novel functions. Nature 423, 185-190.
Fusion cellulose-binding domain of CBP-B domain of protein A
It is a fusion protein between the Clostridium cellulovorans cellulose-binding domain of cellulose-binding protein A (CBPa) with the B domain of staphylococcus aureus protein A (BpA). This fusion protein is used to bind antibodies to cellulose. We use these functions to functionalize a cellulose-based patch into an immunodetection-based system.
Polyhistidine tag (6 His) sequence was added at the 5'end as well as pET43.1a-derived Lac operator, ribosome-binding site (RBS), and T7 terminator.
Cellulose-binding domain of cellulose-binding protein A was taken from iGEM Bielefeld-Germany 2012 team (BBa_K863110) and B domain of protein A was taken from iGEM Warsaw 2008 (BBa_K103003).
Complete CDS of Rat olfactory protein mRNA, clone I7.
CD225 (EpCAM antibody) is commonly used for the test of presence of EpCAM in the laboratories. The sequences of OmpA and TEV protease terminuses are acquired from the Registry.
This gene sequence includes an EpCAM (Epithelial Cell Adhesion Molecule) antigen binding domain, OmpA transmembrane protein (dock) and TEV protease C terminus. It is planned to use this subpart with BBa_772, which contains only TEV protease N terminus differently instead of C terminus, in order to induce a signal reaction in the case of EpCAM presence. EpCAM is an natural antigen which is found high significantly in the cases of some breast and colorectal cancer subtypes. On the upstream region of protein sequence, there are a constitutive pLac promoter and appropiate RBS. To see how this part works: Experience.
There are two protein sequences between domains and the dock: One between the binding domain (antibody) and dock and one between the part of TEV protease.
This BioBrick contains a ''virA'' receptor from ''Agrobacterium tumefaciens'' C58 under the control of a constitutive promotor. We used it to mutate ''virA'' by error-prone PCR. For screening the mutants BBa_K389011 is also needed.
The ''virA'' gene is under the control a medium strong constitutive promotor.
This gene sequence includes both of EpCAM Binding Docks; BBa_K772001 and BBa_K772002. Thus, this composite part is the main component of CTC (Circulating Tumor Cell) detection system. Docks include an EpCAM (Epithelial Cell Adhesion Molecule) antigen binding domain, OmpA transmembrane protein (dock) and TEV protease C or N terminus. Hopefully, after binding to the EpCAM antigen, TEV protease domains will fix together to form the full structure in order to activate the signalling pathway. (For more information: BBa_K772003) EpCAM is an natural antigen which is found high significantly in the cases of some breast and colorectal cancer subtypes. On the upstream region of protein sequences, there are a constitutive pLac promoter and appropriate RBS. To see how this part works: Experience.
There are two protein sequences between domains and the dock: One between the binding domain (antibody) and dock and one between the domain of TEV protease.
cDNA of UV-B sensing protein UVR8 from Arabidopsis thaliana
UVR8 is responsible for UV-B detection in plants. In dark state UVR8 forms a dimer, which breaks into monomers upon exposure to UV-B light (280-315 nm). Later monomer induces transcriptional changes necessary for UV-B damage repairs and protection. In addition a BamHI site is inserted after ATG codon for fusions with other proteins (e.g. tetR-DNA binding domain), to use UVR8 as a UV-B sensing domain.
cDNA of UVR8 from Arabidopsis thaliana was provided by Roman Ulm, University of Geneva.
This gene sequence includes an EpCAM (Epithelial p? Cell Adhesion Molecule) antigen binding domain, OmpA transmembrane protein (dock) and TEV protease N terminus. It is planned to use this subpart with BBa_772, which contains only TEV protease C terminus differently instead of N terminus, in order to induce a signal reaction in the case of EpCAM presence. EpCAM is an natural antigen which is found high significantly in the cases of some breast and colorectal cancer subtypes. On the upstream region of protein sequence, there are a constitutive pLac promoter and appropiate RBS. To see how this part works: Experience.
TNT.R1 has fair affinity (340nM) for trinitrotoluene (TNT)that is not a naturally occurring compound. TNT.R1 is less thermostable than the wildtype receptor. As such, it is suggested that the receptor be used shortly after expression. TNT receptor 1 (TNT.R1) is a computationally-derived ligand receptor specific for TNT. The ribose-binding pocket of ribose-binding protein, a member of the E. coli periplasmic binding protein (PBP) family, was reconfigured so that the receptor recognizes TNT instead of the wild-type ligand.
This part is cloned into BioBrick vector pSB1A2.
Computationally-derived ligand receptor. Looger, L. L., Dwyer, M. A., Smith, J. J. and Hellinga, H. W. (2003). Computational design of receptor and sensor proteins with novel functions. Nature 423: 185 - 190.
This is a gene for the ''virA'' receptor from ''Agrobacterium tumefaciens'' C58. An illegal ''PstI'' restriction site was removed by site-directed mutagenesis.
Illegal ''PstI'' restriction site removed
''Agrobacterium tumefaciens'' C58, TI-plasmid
Background McfR is an organic acid responsive chemoreceptor originally found in***. BBa_K1405004 contains an insulator, RBS sequence, and McfR coding sequence and its expression is under the control of the constitutive promoter BBa_J23100. To be continued.
The chimeric GPCR consists of the N- and C- terminals of the rat OR RI7, flanking the TM2-TM7 ligand-binding domain of the c. elegans OR odr-10. It can be functionally expressed in S. cerevisiae, activating the yeast endogenous MAPK pathway upon binding to the ligand diacetyl.
Digoxigenin is a steroid extracted from the plant Digitalis purpurea and D.lanata. Digoxigenin modified oligonucleotides are widely used as high sensitivity probes for non-radioactive immunoassay s and hybridization experiments. These DIG-probes are monitored by anti-DIG antibodies. These antibodies only show cross-reactivity with blossoms and leaves of D. spec(sole natural sources for DIG) ??? so they are suitable for analysis of any other biological species. Anti-DIG antibodies are either modified with fluorescent dyes (direct detection) or with an enzyme (indirect detection via enzyme-substrate reaction).
Constitutive promoter(J23110), RBS(B0034), NsrR(K2056001), Double terminator(B0015)
EnvZ Signal Peptide and first Transmembrane Domain
For EnvZ fusion protein design. Long description will be added soon.
Source: E. coli EnvZ protein (ncbi: https://www.ncbi.nlm.nih.gov/gene/947272)
protein A from Staphylococcus aureus. Also known as part of Tap-Tag
A composite part comprising of a J23100 promotor and PA2652 translational unit encoding extracellular chemoreceptor
EnvZ Signal Transduction and Kinase Domains
E. coli EnvZ (ncbi: https://www.ncbi.nlm.nih.gov/gene/947272)
EnvZ-LRR chimeric receptor for mDAP recognition
NOD1 (ncbi: https://www.ncbi.nlm.nih.gov/gene/10392)
This antibody was created using mouse hybridomas.
This sequence encodes the amino acid chain of the antigen binding fragment of the TE33 antibody light chain. The antibody is specific for the B subunit of cholera toxin.
Odr-10(diacetyl)GPCR with optimized codon
Junqi kuang, Xihao liao and Jianhui gong
Odr-10(diacetyl)GPCR encodes a diacetyl-sensing G-protein coupled receptor, originating from the C. elegans. If BBa_K1072010 was transformed into the yeast, the pheromone receptors will be actived and coupled to a heterotrimeric G protein, Odr-10. pathway is composedof Gpa1p (Gα), Ste4p (Gβ) and Ste18p (Gγ) subunits.The Gβγ particle transmits the signal to a MAP kinase module and finally follow a inducible promotor FUS1, which will link a reproter, such as GFP, His3 or LacZ.
As discription of most paper, RNA of Odr-10 be used as plate and transform into receptor cells to express member protein successfully.
Conversion of the iGEM parts registry to SBOL2.1