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.
CRY2 and CIB1 are extracted from Arabidopsis thaliana. tCas9 is from GE Share company. VP64 is synthetic construct.
Cph8 synthesize under constitutive promoter.
Constitutive promoter, pTetR: R0040 Cph8: I15010
Renilla (BBa_J52008) with 6X-His tagged for easier protein purification. Sequence was codon optimized for E.coli by IDT optimization tools.
6X-His tagged for easier protein purification. Sequence was codon optimized for E.coli by IDT optimization tools.
Coding sequence of a fused protein (LOV means light, oxygen, and voltage,represent a light sensitive protein domain whereas TAP means tryptophan-activated protein).
LOVTAP sequence contains 2 Pst1 restriction sites
Kindly provided by the authors of : Light-activated DNA binding in a designed allosteric protein; Strickland et al., Proceedings of the National Academy of Sciences (2008) vol. 105 (31) pp. 10709
The CRY2/CIBN interaction is entirely genetically encoded. The binding reverses within minutes in the dark, allowing rapid shutoff of transcription by placing samples in the dark. This fusion protein is for use in LACE(light-activated CRISPR/Cas9 effector) system, and a VP64 fused to its C terminus. To regulate DNA transcription by blue light, the system is based on CRY2/CIBN interaction in which a light-mediated protein interaction brings together two protein (tCas9 and an activation domain VP64) . If we remove the stimulation of blue light, dark reversion of CRY2 will dissociate the interaction with CIBN and shut off transcription.
PompR is a OmpR-controlled promoter which can be positively regulated by phosphorylated OmpR. This promoter is taken from the upstream region of ompC. Phosphorylated OmpR binds to the three operator sites and activates transcription. We insert PompR(BBa_R0082) sequences into the upstream region of our target gene gp35 through restriction-ligation method, which together is connected to vector PSB1C3 afterwards, and therefore we are able to regulate the expression of in through the control of light. Bxb1 gp35 is a serine integrase in Mycobacterium phage Bxb1. This intergrase can exclusively catalyze site-specific recombination. The dismiss of light singal can drive the expression of integrase, which alone can set a DNA sequence(there it means the promoter) flanked by oppositional attB and attP sites, thereby produce an inverted sequence flanked by attL and attR sites. As a consequence, the promotor moves on to express the toxic protein instead of the attractant. We insert the gp35 gene into the downstream region of the PompR promotor by means of 3A synthesis so that the expression of integrase can be regulated by light. In addition, we add a 6N promotor between PompR and gp35 to decrease basal integrase expression above flipping threshold level which could lower the efficiency of int translation. Meanwhile, we insert the recombined sites of attB and attP into the flanking sequences of our constitutive promotor (BBa_J23110 ) and so far we accomplish our reverse promotor system.
we add a 6N promotor between PompR and gp35 to decrease basal integrase expression above flipping threshold level which could lower the efficiency of int translation.
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
Halorhodopsin comes from part BBa_K559000, which is come from genomic sequence of N.pharaonis (genebank ID:J05199.1)
Prokaryotic Cryptochrome 2 (CRY2) ( a blue light stimulated photoreceptor)
Cryptochrome 2 (CRY2) is a blue light stimulated photoreceptor, when exposed to blue light, it would interact withthe N-terminal fragment of CIB1 (CIBN) . This part is full-length CRY2 CDS. Optogenetic systems enable precise spatial and temporal control of cell behavior. A light-activated CRISPR/Cas9 effector (LACE) system that induces transcription of endogenous genes in the presence of blue light.This was accomplished by fusing the light-inducible heterodimerizing proteins CRY2 and CIB1 to a transactivation domain and the catalytically inactive tCas9, respectively. The versatile LACE system can be easily directed to new DNA sequences for the dynamic regulation of endogenous genes.
mutant EcoR 1 : TGTTGGGAATTc--->TGTTGGGAATTg
The light-inducible heterodimerizering proteins CRY2 and CIB1 are both from Arabidopsis thaliana.
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.
Halorhodopsin is an inward-directed light-driven chloride ion pump originating from Halobacterium. It utilizes light to pump chloride ions against chloride concentration difference into cells from the environment. We added a constitutive working and regulatable pLac promoter in the upstream region of the part in order to use it in our design easily.
Please refer to http://partsregistry.org/Part:BBa_K559000:Design for more detail about halorhodopsin. Please refer to http://partsregistry.org/Part:BBa_J04500:Design for more detail about pLac promoter.
Please refer to http://partsregistry.org/Part:BBa_K559000:Design, both of the genes are gathered from the Registry distribution.
Engineered green fluorescent protein that modulates its fluorescence in response to changes in calcium ion (Ca2+) concentration.
Coding sequence of a fused protein (LOV means light, oxygen, and voltage,represent a light sensitive protein domain whereas TAP means tryptophan-activated protein) followed by a terminator.
LOVTap sequence contains 2 Pst1 restriction sites
Eukaryotic Cryptochrome 2 (CRY2) ( a blue light stimulated photoreceptor)
Cryptochrome 2 (CRY2) is a blue light stimulated photoreceptor, when exposed to blue light, it would interact withthe N-terminal fragment of CIB1 (CIBN) . This part is full-length CRY2 CDS. Optogenetic systems enable precise spatial and temporal control of cell behavior. A light-activated CRISPR/Cas9 effector (LACE) system that induces transcription of endogenous genes in the presence of blue light.This was accomplished by fusing the light-inducible heterodimerizing proteins CRY2 and CIB1 to a transactivation domain and the catalytically inactive tCas9, respectively. The versatile LACE system can be easily directed to new DNA sequences for the dynamic regulation of endogenous genes[1].
This is a coding sequence for Pif3 gene with BD ends for Aar1 cloning. Binds with PhyB at 720nm light and unbinds at 660nm.
As a BD part, there is no ATG, and the stop codon is included. For more information on AarI cloning, please refer to the UCSF 2008 igem page.
Complete coding sequence for fused protein LOVTAP, with inducible promoter, RBS site and terminator.
In order to work,Cph8 has to form chromophore with PCB biosynthetic genes, where the formation of PCB requires the gene pcyA and hol to function as accounted below. Hol is a sort of Iron red pigment oxidase which can oxidize the heme group using a ferredoxin cofactor, generating biliverdin IXalpha. And then, PcyA, a kind of ferredoxin oxidase from Synechocystis Sauv, functions to turn biliverdin IXalpha (BV) into PCB. We connected gene pcyA（BBa_I15009）and ho1（BBa_I15008）together along with the constitutive promoter(BBa_J23100) to ensure a continuous work of the photoinduction system in our E.coli.
We used oe-pcr to assemble these parts together seamlessly.
pcyA gene and ho1 gene both come from Synechocystis.
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
Halorhodopsin come from N.pharaonis, gene bank J05199.1
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
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.
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.
This is the light sensor Cph8 with a promoter and RBS.
This Halorhodopsin gene allow the photoreceptor protein be expressed, to regulate the chloride ion pumped in by light intensity
The DNA sequence selected from ID J05199.1 of 61nt to 700nt for protein coding
It is from N.pharaonis, gene back ID J05199.1
Engineered blue fluorescent protein that modulates its fluorescence in response to changes in calcium ion (Ca2+) concentration.
pcyA converts biliverdin IXalpha (BV) to phycocyanobilin (PCB). Strong RBS (efficiency=0.6).
Chimeric Cph1 light receptor/EnvZ protein.
Silent mutation at base 108 (G-A) to remove PstI site
See part BBa_K191005 for information on fused protein LOVTAP. This part is a mutated version of LOVTAP: amino acid #427 ILE is mutated into PHE
This is a coding sequence for Pif3 gene with AB ends for Aar1 cloning. Binds with PhyB at 720nm light and unbinds at 660nm. Fuses only at the N-terminal only.
As a BD part, there is no ATG, and the stop codon is included. For more information on AarI cloning, please refer to the UCSF 2008 iGEM page. Fuses only at the N-terminal only.
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.
This composite part contains a promoter and ribosome-binding-site, a gene for ZntR, a terminator, and a promoter for ZntA. In the absence of Zinc, the ZntR protein binds to the ZntA binding site. In the presence of zinc, ZntR undergoes a conformation, does not attach to the binding site, and allows for transcription of ZntA. Essentially, this part acts as a sensor for the presence of zinc.
We had to enter silent mutations to change the DNA sequence without affecting the amino acid sequence because there were palindromes greater than 10 bp in every basic part.
The genomic sequence comes from parts of an E. Coli protein (ZntR).
One of two requisite genes required for the biosynthesis of phycocyanobilin from heme.
phycocyanobilin:ferredoxin oxidoreductase (PcyA) from synechocystis
Second of two required phycocyanobilin biosynthetic genes.
Promoter LacI - RBS - LovTAP(ILE427PHE) - Term
Complete coding sequence for mutated protein LOVTAP (mutation site: a.a #427 ILE is changed into PHE), with inducible promoter, RBS site and terminator.
Site specific mutation on plasmid with part BBa_K191003
Lov-HTH (light sensor with Ptet repression) light->PtetO repressed
This page is about a light sensor and regulator protein: Lov-HTH. Lov-HTH is a artificial light sensor designed by iGEM12 Fudan Lux team. This protein include two function domain, Lov the light sensing domain and HTH the DNA binding domain.
The HTH motif swap is the main issue in our design. We carefully choose the HTH sequence and swap it into the original amino acid sequence.
The sequence is reverse translated from the protein EL222 http://www.rcsb.org/pdb/explore/explore.do?structureId=3P7N which is from Erythrobacter litoralis. The DNA is synthesized by Invitrogen company.
Encodes the N-terminal fragment of transcription factor CIB1 (cryptochrome-interacting basic-helix-loop-helix). CIB1 interacts with CRY2 (cryptochrome 2) in a blue light-specific manner in yeast and Arabidopsis cells, and it acts together with additional CIB1-related proteins to promote CRY2-dependent floral initiation. CIB1 positively regulates FT expression. Cryptochrome 2 (CRY2) is a blue light stimulated photoreceptor, when exposed to blue light, it would interact withthe N-terminal fragment of CIB1 (CIBN) . Optogenetic systems enable precise spatial and temporal control of cell behavior. A light-activated CRISPR/Cas9 effector (LACE) system that induces transcription of endogenous genes in the presence of blue light.This was accomplished by fusing the light-inducible heterodimerizing proteins CRY2 and CIB1 to a transactivation domain and the catalytically inactive tCas9, respectively. The versatile LACE system can be easily directed to new DNA sequences for the dynamic regulation of endogenous genes[1].
Lov-tetR (light sensor with Ptet repression) light->PtetO repressed
This part code a protein as a light sensor and repressor. With the existence of this protein, expression downstream the PtetO promoter will be repressed under the 450nm blue light. This protein, which is actually a well designed fusion protein, include two domain. The Lov domain is from Avena sativa phototrophin1(AsLOV2). The tetR is from the tetracycline resistance, which could combine the tetracycline and enable the expression of the tetA. We carefully designed the linker between these two parts, and fulfilled our sensor part in our biowave circuit.
This part is designed for our biowave project.
This coding sequence is form the Avena sativa phototrophin1(AsLOV2) and tetracycline resistance. The whole gene is synthesized by Invitrogen company.
This photoreceptor was assembled by Strickland et al., and consists of a LOV (light-oxygen-voltage) domain of Avena sativa phototropin1 (AsLOV2) that senses blue light, fused to the trpR- DNA binding domain of the transcription factor trp repressor. The resulting protein is named LovTAP: LOV- and tryptophan-activated protein. LOV domains bind a flavin-mononucleotide (FMN) or flavin-adenine-dinucleotide (FAD) cofactor, which are used in a wide variety of metabolic pathways as cofactors in redox reactions and are available in most organisms. The cofactor has a broad absorption spectrum, with a maximum at 450 nm. Besides, the core of the LOV domain is often flanked by amino- or carboxy-terminal helices, termed A???α and Jα, respectively In the LovTAP construction, AsLOV2 domain via its carboxyl-terminal Jα ???helix was ligated to an amino-terminal truncation of TrpR. The resulting protein has a domain-domain overlap with a shared helix. Thus, photoexcitation would change the conformation of the protein, in turn changing the stability of the shared-helix-domain contacts. Under the presence of light, absorption of a photon leads to the formation of a covalent adduct between the flavin mononucleotide (FMN) cofactor and a conserved cysteine residue in the AsLOV2 domain, which results in conformational rearrangements in LovTAP. This change impacts the affinity of the shared helix for the two domains: disrupting the contacts between the shared helix and the LOV domain and enabling the association of the shared helix with the TrpR domain, which establishes DNA-binding affinity and LovTAP can then bind DNA as an homodimer, repressing the transcription of the genes downstream of its binding sites. In the dark, when the shared helix contacts the LOV domain, the TrpR domain's DNA-binding affinity decreases and LovTAP is in an inactive conformation.
We decided to synthesize a new LovTAP part, that in comparison with the Part:BBa_K191003 that is already at the registry, has the following differences: 1. The 2 PstI restriction sites were removed from the coding region of LovTap. 2. We included a punctual mutation to change the ILE427 by a PHE427, as was proposed by the model results of the team iGEM09_EPF-Lausanne. With this mutation LovTAP should react faster and the conformational change should be more stable (the protein stays in the active form for longer, under light induction). 3. The part does not include a promoter. We eliminated the inverting regulator sensitive to LacI and CAP protein(Part:BBa_R0010). According to the report of EPF-Laussane team, the expression levels of LovTAP under the inverting regulator, doesn??t seem to show differences to the induction with IPTG. Thus, we decided to remove it and as we know that the level expression of LovTAP must be low (because at high levels of expresion, there is unspecific regulation under darkness and light states), we plan to test weak promoters to choose the best option and include it in the construction. 4. The RBS was changed from a strong (Part: BBa_B0030) to a medium strength (Part:BBa_B0032), thus expecting lower levels of lovTAP protein. 5. The stop codon tga was changed for two taa.
Chiameric protein assembled by Strickland et al. Reference: Strickland, D., Moffat, K., & Sosnick, T. (2008). Light-activated DNA binding in a designed allosteric protein. Proceedings of the National Academy of Sciences, 105(31), 10709. National Acad Sciences. Retrieved from http://www.pnas.org/content/105/31/10709.full.
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.
Engineered red fluorescent protein that modulates its fluorescence in response to changes in calcium ion (Ca2+) concentration.