Trz hybrid signal transduction protein: this was created by fusing the extracellular receptor domain of the chemoreceptor Trg with the intracellular signal transduction domain of 2-component sensor protein EnvZ, which normally phosphorylates response regulator OmpR (see Baumgartner, J.W., Kim, C., Brissette, R.E., Inouye, M., Park, C., and Hazelbauer, G.L. 1994. Transmembrane signalling by a hybrid protein: communication from the domain of chemoreceptor Trg that recognizes sugar-binding proteins to the kinase/phosphatase domain of the osmosensor EnvZ. J. Bacteriol. 176, 1157-1163. This part can activate genes attached to an OmpR-responsive promoter such as PompC. To avoid inappropriate activation via EnvZ, it may be necessary to use a chassis in which EnvZ is not present.
Kindly supplied by Prof. G. Hazelbauer, and BioBricked by the University of Edinburgh iGEM team 2009.
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.
CPX Terminal Surface Display Protein with Polystyrene-Binding Peptide
Rice JJ, Schohn A, Bessette PH, Boulware KT, and Daugherty PS. Bacterial display using circularly permuted outer membrane protein OmpX yields high affinity peptide ligands. Protein Sci 2006 Apr; 15(4) 825-36. doi:10.1110/ps.051897806 pmid:16600968
dnMyD88 is a dominant negative form of MyD88, which has an important role in TLR signalling pathway. dnMyD88 contains only TIR domain of MyD88.
malate chemorecptor from Pseudomonas aurenginosa
genomic sequence from Pseudomonas aurenginosa strain PA01
Tar expression system (promoter+RBS+coding+terminator)
introduction Tar chemoreceptor is one of four native chemoreceptors of E.coli, it is mediate chemotaxis response towards aspartate and away from Ni and Co (1). This device allows the expression of a functionally native Tar chemoreceptor. usage and biology This device constructed from strong promoter (J23100) and RBS (B0034) ensuring high expression of Tar chemoreceptor. High expression of a sole chemoreceptor increase the sensitivity of the bacteria to the substances that interacts with this receptor (מקור). This property makes the system efficient and useful even in low concentrations of desired materials. expriments and results In order to test the device it was cloned to chemoreceptor free strain (UU1250) and tested for chemotaxis ability using swarming assay. As can be seen in figure 1 the cells expressing the Tar chemoreceptor create a halo indicating a functional chemotaxis result, which compared to the negative and positive controls. The halo can be seen in a matter of hours ensuring that the halo is a result of a chemotaxis response. Figure 1 Fig1. (a) Tar expression in UU1250 strain, resulting a halo indicating a functional chemotaxis response. (b) Negative control- UU1250 strain w/o the Tar expression plasmid. (c) positive control - ΔZ strain expressing all chemoreceptors. The part was successfully sequenced (fig2) ensuring the present of the promoter, RBS, Tar receptor and terminator. Figure 2 Fig 2. (a) Sequencing chromatogram of the K1992004 BioBrick- the RBS is marked. (b) The alignment of the sequencing results in compared to the designed sequence - alignment was done using snapgene software.
pGAL1 + Flag + odr-10 + GFP + ADH1Te + pFUS1 + BFP + ADH1Te
Tar is a transmembrane receptor in E. coli which mediates chemotaxis towards aspartate.
The promoter of tar is replaced by lacI promoter and the RBS (Shine-Dalgarno sequence) is removed to reduce the expression level. As it turned out, lower level of expression ensured better chemotaxis.
The sequence is cloned from the plasmid pPD12, a kind gift from Dr. Goulian.
membrane progesterone receptor (Xenopus laevis)
Membrane progesterone receptor of Xenopus laevis. Upon ligand binding, the receptor represses expression of Fet3 in yeast.
restriction sites removed and codon usage optimized for yeast
Toll-Like Receptor 3 transmembrane region and cytoplasmic domain, with stop codon added.
amplified from TLR3 gene, obtained from Invivogen, plasmid pUNO-hTLR3
VirA/G signaling system + sensitivity tuner + luciferase
This part combines a complete VirA/G signaling system with a sensitivity tuner before a luciferase gene under the control of a ''vir'' promoter. The sensitivity tuner enhances a signal from a promoter so it enhances the readout luciferase.
sensitivity tuner to enhance the luciferase signal
* luciferase from pGL4.10luc2 (Promega) * sensitivity tuner part, terminators and constitutive promoter from the partsregistry * ''virG'' was synthesized by Mr. Gene * ''virA'' and ''vir'' promoter from ''Agrobacterium tumefaciens'' C58, TI-plasmid
HasR is the membrane-bound receptor in the Has system, a heme acquisition system originally from the Serratia marcescens. The HasR protein is involved in binding with a heme-loaded HasA hemophore to trigger downstream HasS and HasI. It has been shown that the N terminal of HasR is especially crucial for signal transduction. With the heme-loaded hasA bound to hasR, it induces a conformational change in HasR that causes the activation of HasS, an anti-sigma factor. HasS then releases HasI (sigma factor) that was initially bound to it and HasI ultimately is able to trigger expression of genes under control of pHas (part number).
The HasR gene is originally found in the Serratia marcescens. We obtained the gene sequence from the National Center for Biotechnology Information (NCBI) database (http://www.ncbi.nlm.nih.gov/nuccore/X81195.2) and synthesised the gene.
Stephen Lee, Roshan Kumar, Joe Levine, Ziyan Chu (Polkadorks, IAP 2004)
Coding sequence for Tar-EnvZ fusion protein. Tar is the bacterial chemotaxis receptor for aspartate. EnvZ is an osmosensor kinase, which, through phosphorylation of OmpR, regulates transcription of porin genes. This fusion protein combines the extracellular and transmembrane domains of Tar with the intracellular domain of EnvZ. The fusion converts chemical binding of aspartate to production of transcription factors.
A silent point mutation was made at base 423 (A -> C) to eliminate an XbaI restriction enzyme site. The mutation was designed to match codons with similar frequencies of usage in E. coli.
This plasmid contains code for pBAD promoter, AIP receiver Agr A and Agr C, GFP along with double terminator.
Sequence was cloned using BBa_E0240 and BBa_I746103 from iGEM parts registry.
fhuA - Outer membrane transporter for ferrichrome-iron
fhuA is an outer membrane (OM) protein that is found in the gram negative bacterium, E.coli. It is involved in the active-transport (energy-dependent uptake) of ferrichrome-iron from the extracellular environment. However fhuA is only able to bind ferrichrome-iron (i.e. ferrichrome iron is a ligand of fhuA protein). For the uptake to be completed fhuA needs to form a complex with another protein, tonB that carries out the energy-dependent uptake by using ATP as substrate to undergo the relevant conformational changes. This biobrick ONLY encodes for fhuA. fhuA is a beta-barell protein (22 anti-parallel beta-strands) and is composed of 747 aminoacids. This receptor protein may be used as an attachment point by some colicins. References: Locher K.P., Rees B., Koebnik R., Mitschler A., Moulinier L., Rosenusch J.P. and Moras D. (1998),Transmembrane signalling across the Ligand-Gated FhuAReceptor: Crystal structures of Free and Ferrichrome-Bound States reveal allosteric changes. Cell 95,771-778. Uniprot access number: P06971 Ecogene: http://ecogene.org/geneInfo.php?eg_id=EG10302
The coding sequence (CDS) was already in compliance with assembly standard 10 (RFC10).
fhuA is part of the chromosomal genome of E.coli. In particular it can be found at 3.61min and follows the clockwise direction.
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.
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.
EnvZ Signal Transduction and Kinase Domains
E. coli EnvZ (ncbi: https://www.ncbi.nlm.nih.gov/gene/947272)
The rpfC gene from Xanthomonas campestris
This is the coding sequence for the RpfC(regulation of pathogenicity factor C) protein. It is the sensor in the two-component signal transduction of Xcc that activates the regulator RpfG(Bba_K1315003) in the presence of DSF.
The sequence had 1 EcoRI site and 3 PstI sites that were all mutated out by site-directed mutagenesis.
The source of this part is ''Xanthomonas campestris pv. campestris''(Xcc) genomic DNA which was provided by the Robert Ryan lab in the Molecular Microbiology department of the University of Dundee, UK.
EnvZ-LRR chimeric receptor for mDAP recognition
Coding sequence for Homo sapien Cripto-1 membrane protein.
Ryan Kawalerski, Tasnia Islam, Joshua Hombrebueno, Sarah Heacox, Samara Khan, Daniel Thach, Meghan Bialt deCelie.
YtvA is a blue light receptor endogenously found in ''B.subtilis''. Activation of the YtvA light receptor activates the mild stress response pathway, accumulating in sigma B activation. Previous research has shown that sigma B dependent transcription in response to blue light light exposure can be enhanced by the over expression of the YtvA receptor 1. We over expressed YtvA to enhance the transcriptional output of sigma B promoters in response to blue light.
Biobrick standard was applied to the YtvA sequence.
The part was designed using the sequence from the B.subtilis genome. This sequence was then synthesised by Geneart.
This BioBrick is for testing the virA/G signaling system. The read-out is the luciferase <partinfo>K389004</partinfo>. This system contains an unmutated ''virA'', so it is possible to measure, how the natural virA/G system works and reacts.
this is only for measuring the natural virA/G system
''A. tumefaciens'' C58 TI-plasmid, partsregistry, Mr. Gene, Promega's pGL4.10luc2 plasmid
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.
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.
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.
Molecular mechanism of the photosensor The fusion,chimera-protein coupled to the chemotaxis pathway. Figure taken from Trivedi et al.(2) The fusion,chimera-protein coupled to the chemotaxis pathway. Figure taken from Trivedi et al.(2) The photosensor acts directly on the tumbling frequency by effecting E. Coli's normal chemotaxis pathway. When exposed to bluelight, the sensory rhodopsin II will absorb the photons and undergo a change in ultrastructure, which is being transduced through HtrII on to the Tar domain. This effects decreases the autophosphorylation of the CheA protein, which in turn again decreases the amount of phosphorylated CheY, which just means that less of it will get phosphorylated. If there is less of the CheY-P, then there is a smaller chance of one of these molecules binding to the flagellar motor and making it turn clockwise, thereby inducing a lowered tumbling frequency in the system. The photosensor can also act in the opposite way, inducing a higher tumbling rate in the bacteria. Which effect the sensor will have depends on where NpHtrII and StTar are fused in the HAMP domain. If the fusion contains 20 more basepair of the HtrII domain and 20 less of the Tar domain, the photosensor would have the opposite effect and would be increasing the autophosphorylation of CheA. Usage and parameters The part requires retinal to work in E.Coli. This can be achieved through adding retinal to the liquid growth medium and/or the plates. Currently we are doing experiments on wether the part also functions with an internal retinal source, ie retinal synthesis in E. Coli. On top of adding retinal the cells will have to be grown in the dark for at least two hours after the addition of retinal to the growth medium if you want to see an effect. This is so that the photosensor is not exposed to light before the experiments and will result in maximum output if exposed to blue light. Results Bacteria containing this part will exhibit a lowered tumbling rate when exposed to blue light (wavelengths around 350nm - 450nm). This was analysed with the help of video microscopy and the open source software "CellTrack". The individual cells trajectory was tracked and their speed measured. The tracking results are as follows: From left to right, trajectory of: E.Coli with photosensor exposed to blue light, E.Coli with photosensor exposed to red light and E.Coli Mg1655 Wildtype exposed to blue light: (Blue dots show the location of the cell in the given frame, so the number of dots equals the number of frames from the sample.) The phototaxic bacteria move more in a straight line when exposed to bluelight, as can be seen when comparing the trajectories of the thee bacteria given earlier. These were taken from a batch of 10 cells tracked per sample.
Design Notes NpSopII-NpHtrII-StTar fusion-chimera protein sequence from Jung K-H, Spudich EN, Trivedi VD and Spudich JL (1). The first 224 amino acid residues come from the NpSopII gene, encoding a bluelight photon receptor with 15 residues removed at the C-terminal end. The following 9 amino acids are a linker. the last part is HtrII fused with Tsr from E.Coli at the HAMP domain. The complex' first 125 amino acid residues come from HtrII and the remaining 279 from Tsr. The constitutive promoter R0040 was chosen for its medium strength, so that there would be a rather large amount of the fusion, chimera protein expressed, but not so much that it would be lethal to the cell. The easy repression and inhibition of repression through TetR and tetracycline makes for an easily controlled expression if that is needed. The doible terminator B0015 was chosen for it's reliability and good reviews on the partsregistry.
Source Sensory Rhodopsin II and HtrII domains were taken from the genome of Natronomonas Pharaonis. The Tar methyl accepting chemotaxis protein was taken from Salmonella Enterica Serovar Typhimurium. The synthetic linker and fusing of the HAMP domains of HtrII and StTar was done by the team behind the referenced article (1).
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)
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.
Fibroblast Growth Factor-1 Receptor Coding Sequence
The Fibroblast Growth Factor-1 Receptor (FGFR-1) is a receptor that is commonly found in melanocytes and is involved with melanocyte differentiation, migration, and division. Malignant melanoma is characterized by abnormally high ligand expression of Fibroblast Growth Factor-2 (FGF2), otherwise known as the Basic Fibroblast Growth Factor (BFGF). The FGFR-1 receptor as a high affinity towards binding to the FGF2 ligand present upon the surface of potentially malignant melanocytes, and therefore may be utilized to detect presence of malignant melanoma tumor.
The start codon sequence has been added to the FGFR-1 coding sequence.
The part has been derived from a genomic sequence provisioned by the Ensembl Database.
Tar native RBS expression system (promoter+RBS+coding+terminator)
introduction Tar chemoreceptor is one of four native chemoreceptors of E.coli, it is mediate chemotaxis response towards aspartate and away from Ni and Co (מקור). This device allows the expression of a functionally native Tar chemoreceptor. usage and biology This device is very similar to our K1992004 device the only difference is the RBS, here the expression system is using the native RBS of the Tar receptor as found in the E.coli genome (K1992000). expriments and results In order to test the device it was cloned to chemoreceptor free strain (UU1250) and tested for chemotaxis ability using swarming assay. As can be seen in figure 1 the cells expressing the Tar chemoreceptor create a halo indicating a functional chemotaxis result, which compared to the Tar expression system using a strong RBS(B0034). Figure 1 Fig1. (a) Tar expression in UU1250 strain cloned with K1992004 expretion system - strong RBS. (b) Tar expression in UU1250 strain cloned with K1992004 expretion system - Tar native RBS The part was successfully sequenced (fig2) ensuring the present of the promoter, RBS, Tar receptor and terminator. Figure 2 Fig 2. (a) Sequencing chromatogram of the K1992005 BioBrick- the RBS is marked. (b) The alignment of the sequencing results in compared to the designed sequence - alignment was done using snapgene software.
toxicity-gene activator from Vibrio cholerae
shortened BBa_J07008. Typical ToxR-periplasm-region subsitutions are done @ aa 211. explanation of ToxR domains @ http://model.mit.edu/igem/images/d/d7/Kolmar95.pdf ELIMINATE XbaI SITE PLZZZZZ
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.
This is a mutant version of E. Coli Tar receptor and changes the chemotaxis properties. This receptor now responds to phenylacetic acid (PAA).
LacI promoter without Shine-Dalgarno sequence ensures sensitive and specific chemotaxis
VirA/G signaling system without read-out
This BioBrick is a virA/G signaling system without read-out. It contains an unmutated ''virA'' gene and a ''virG'' gene that works without ''rpoA'' from ''A. tumefaciens'' and a ''vir'' promoter.
This BioBrick can be used to control any gene with a ''vir'' promoter
''A. tumefaciens'' C58 TI-plasmid, partsregistry, Mr. Gene
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
AgrC protein coding sequence. AgrC is main component of S.aureus agr quorum sensing system.
AgrC is a transmembrane protein, which is the sensor molecule of a typical two-component signal system in S.aureus. AgrC possesses several key amino acid motifs typical of histidine protein kinase sensor. The AgrC sensor kinase can specifically binds to corresponding AIP, which secreted only from specific S.aureus, and the composite of AgrC and AIP then leads to phosphorylation of AgrA. AgrA in its phosphorylated sate activates transcription from both P2 and P3, leading to the production of GFP and 3OC6HSL. Thus we can detect the presence of S.aureus expediently by observing the expression of GFP.
we employed site-specific mutagenesis(AgrC-I A101T) to replace a amino acid residue in AgrC-I so that the mutated AgrC can bound to both AIP-I and AIP-IV.
Toll-Like Receptor 9 transmembrane region and cytoplasmic domain.
amplified from TLR9 gene, obtained from Invivogen, plasmid pUNO-hTLR9
lamB is the a porin protein which is part of the maltose pathway. It is also the receptor associated and required fro lambda phage infection.
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.
OmpA is derived from the genomic DNA of BL21 (DE3) E. Coli strain while SH3 is from human Grb2.
pGAL1 + Flag + Brho + odr-10 + GFP + ADH1Te + pFUS1 + BFP + ADH1Te
NOD1 (ncbi: https://www.ncbi.nlm.nih.gov/gene/10392)
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.
Tetracycline repressor DNA binding domain fusion with truncated version of UVR8 (tetR-DBD-UVR8). UVR8 is a UV-B sensing receptor in plants necessary for UV-B protection. In dark state UVR8 forms a dimer which is monomerized by the UV-B light (280-315 nm). A truncated version of UVR8 (14-440 amino acids) was fused with tetR DNA binding domain (tetR-DBD), lacking of dimerization domain. Such mutant shows no repression of Ptet promoter, however fusion with dimerizing protein (UVR8) returns tetR-DBD activity. Later UV-B exposure breaks UVR8 dimer and releases tetR-DBD-UVR8 from Ptet, activating transcription. Thus, this tetR-DBD-UVR8 can be used as a one step UV-B on switch.
Contains two illegal PstI sites in UVR8 coding region and BamHI site was used for protein fusion
tetR-DBD was cloned from E.coli tetracyclin repressor tetR from transposon Tn10 cDNA of UVR8 was provided by Roman Ulm, University of Geneva.
Toll-Like Receptor 9 cytoplasmic domain. Cloned into pSB1AK3 vector.
PAA receptor weakened expression cassette
Chemokine (C-C motif) receptor 5, a HIV-1 co-receptor.
The part is cloned into BBa_J52017, a vector with eukaryontic terminator. PstI and EcoRI restriction sites inside CCR5 gene were mutated.
This part combines a complete VirA/G signaling system with a sensitivity tuner before a luciferase gene under the control of a vir promoter. The sensitivity tuner enhances a signal from a promoter so it enhances the readout luciferase.
* luciferase from pGL4.10luc2 (Promega) * sensitivity tuner part, terminators and constitutive promoter from the partsregistry * ''virG'' was synthesized by Mr. Gene * ''virA'' and ''vir'' promoter from Agrobacterium tumefaciens C58, TI-plasmid
Toll-like receptor 3 cytoplasmic domain.
Cloned into pSBAK3 vector with terminator. Start codon is NOT included.
Strong promoter BBa_J23119. Chimeric Red light receptor cph8 BBa_I15010 (cph1-envZ) and proteins involved in phycobilin biosynthesis ho1 (BBa_I15008), pcyA (BBa_I15009).
Sequences are from Cyanobacteria : - Cph1 domain - ho1 BBa_I15008 - pcyA BBa_I15009
fiu ??? Outer Membrane Ferric Iron Transporter
===Biology of Fiu=== The fiu gene encodes for an outer membrane transporter found in E.coli, a gram negative bacterium. It is involved in the active transport (energy dependent uptake) of ferric iron from the extracellular environment. This outer membrane receptor can only bind ferric iron and is a catecholate siderophore. For uptake into the cell to be completed fiu has to complex with another protein known as TonB (a transperiplasmic protein) that will carry out the active transport by using ATP as an energy source. ===What this Biobrick gives you upon expression=== This biobrick encodes ONLY for fiu. ===Fiu Statistics=== Fiu belongs to the family of Ton-B dependent receptors and is related to fhuA, hence it also has a beta-barrel structure. {| style="color:white; background-color:#0066CC;" cellpadding="20" cellspacing="0" border="2" !Chassis !Monomer/Multimer !Aminoacid Length/Weight !Localisation !Structure |- |E.Coli |Monomer |760/81.96kDa |Outer Membrane/OMV |beta-barrel |} ===References=== [http://ecoliwiki.net/colipedia/index.php/fiu:Gene_Product%28s%29#Domains.2FMotifs.2FModification_Sites E.Coli wiki net] Riley, M. et al. (2006) Nucleic Acids Res 34:1-6 (corrected supplemental data from B. Wanner) [http://www.ecogene.org/geneInfo.php?eg_id=EG13317 Ecogene number EG13317] [http://www.uniprot.org/uniprot/P75780 UniProt Accession number P75780]
In order to comply with assembly standard 10 (RFC10) the stop codon for fiu had to be changed to TAA.
Fiu gene is part of the chromosomal E.coli genome. In particular it is found at 18.07min with orientation in the counter-clockwise direction. [http://www.ecogene.org/geneInfo.php?eg_id=EG13317 Further details at EcoGene]
The Fibroblast Growth Factor Receptor-1 and Fibroblast Growth Factor Receptor Substrate 2 complex comprises a fusion protein complex that has the capability to mediate signal transduction. In melanocytes, this multi-protein complex mediate signal transduction to such pathways as the MAPK pathway, which regulate melanocyte proliferation, migration, and differentiation.
No specific design considerations were dealt with.
The FGFR-1 protein sequence has been derived from the Emblem Database. The FRS2 protein sequence has been derived from the UnitProt Database.
Chemokine (CXC motif) receptor 4, fused to N-terminal half of ubiquitin.
CXCR4 and NUb fragments were fused with PCR overlap extension method without any linker added. The part is cloned into BBa_J52017 (a vector with eukaryontic terminator).
CXCR4 gene obtained from Origene, NUb gene obtained from Dualsystems.
Fusion protein Trg-EnvZ for signal transduction
We created a hybrid gene coding for a protein in which Trg (chemoreceptor) contributed its periplasmic and transmembrane domains as well as a short cytoplasmic segment and EnvZ (osmosensor) contributed its cytoplasmic kinase/phosphatase domain.
This part is identical to BBa_I15010, the cph8 red light sensor. We were unable to transform BBa_I15010 from the the 2011 iGEM distribution kit. PCR attempts using VF2 and VR failed to amplify any DNA from the well sample. In the end, we chose to amplify cph8 from Jeffrey Tabor's pJT122 plasmid provided by Chris Voigt. The cph8 in pJT122 contained an internal PstI site, which was removed by point-mutagenesis. The resultant DNA sequence is identical to that of BBa_I15010.
The source DNA (pJT122) contained illegal restriction site (PstI) that had to be removed.
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.
OmpA-HIV protease site-SH3 expression cassette
EM7 is a synthesized promoter based on the consensus sequence of E. Coli promoter.
OmpA-HIV protease site-SH3 from BBa_K533002 EM7 promoter is synthesized.
Chimeric Cph1 light receptor/EnvZ protein.
Silent mutation at base 108 (G-A) to remove PstI site
membrane progesterone receptor (Danio rerio)
Membrane progesterone receptor of Danio rerio. Upon ligand binding, the receptor represses expression of Fet3 in yeast.
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.
amplified from TLR4 gene, obtained from Invivogen, plasmid pUNO-hTLR4
This BioBrick is for testing the virA/G signaling system. The read-out is the mRFP <partinfo>E1010</partinfo>. This system contains an unmutated ''virA'', so it is possible to measure, how the natural virA/G system works and reacts.
==What this part gives upon expression== This part encodes for FhuA directly as it is already in frame with a pBAD/Arabinose regulated promoter. Promoter Induction can occur with 0.1% Arabinose in the culture environment. FhuA is an E.coli Outer Membrane (OM) protein used for scavenging iron from the nutrient medium. For more information on FhuA visit the FhuA main page (<partinfo>K259000</partinfo>). ==Potential uses of this Device== This part can be used also to create protein fusions by adding other proteins to it's 3' end. Protein fusion can be accomplished straightforwardly using the Bioscaffold-Linker biobricks <partinfo>K259003</partinfo>. Protein fusions with FhuA may allow delivery of proteins to the OM of E.coli cells or inclusion into Outer Membrane Vesicles (see iGEM'09 Bristol-BCCS project).
The part was assembled using standard RFC10 assembly.
The arabinose promoter occurs naturally in E.coli. The RBS is designed from the Anderson family. FhuA also occurs naturally in E.coli.
This is the light sensor Cph8 with a promoter and RBS.
Synthetic periplasmic binding protein that docks a vanillin molecule
Chemokine (C-C motif) receptor 5, fused to N-terminal half of ubiquitin.
PstI and EcoRI restriction sites inside CCR5 gene were mutated without changing the aminoacid sequence. CCR5 and NUb fragments were fused with PCR overlap extension method. The part is cloned into BBa_J52017, a vector with eukaryontic terminator.
CCR5 gene obtained from Origene, NUb gene obtained from Dualsystems.