Acyltransferase Candidate 1 Expression Construct
MAS is a plant specific promoter which is constitutively expressed. This part is compatible with the GoldenGate MoClo Assembly Standard as it is free from BsaI and BpiI recognition sequences.
Domestication for GoldenGate by removal of BsaI and BpiI recognition sequences.
35S:LexABD-DronpaK:T35S - 35S:DronpaN-VP16:T35S
Composite composed of the two transcriptional units needed to obtain a toggle switch control by violet and cian light.
Acyltransferase Candidate 3 -Fused to GFP -Expression Construct
Acylates starch with GFP tag for visualisation of localisation
GoldenGate compatible Bax - cell death inducer
BAX coding sequence (Bcl-2-associated X) protein, a pro-apoptotic member of the Bcl-2 protein family which induces cell death. The part has been made compatible with the GoldenGate MoClo Assembly Standard and is free from BsaI and BpiI recognition sequences. Codon-optimised for expression in dicotyledenous plants.
Domestication for GoldenGate by removal of internal BsaI and BpiI recognition sequences. Codon-optimised for expression in dicotyledenous plants.
35s:5'+ Ga20ox consense + SAGTI-Luciferase + Tnos
Modul of the gRNA testing system created by Valencia UPV 2016 team. This system aims to test the functionality and efficiency of a chosen gRNA to target a particular gene. This system is modular, as the gene inserted can be chosen by the user, only taking into account the required overhangs (5'-AATG-3' and 5'-TTCG-3') It includes a promoter 35s with a 5' region (BBa_K2017002), 20 nucleotides of the Ga20ox consensus gene of Oryza sativa (rice), the reporter luciferase with SAGTI linker and the terminator Tnos. Luciferase is out of its reading frame due to a nucleotide added upstream to the linker, and the initial ATG has been removed. This means that when the luciferase is translated, it will not be functional. To make it functional, it is necessary to make indels in the Ga20ox fragment that put the luciferase in the correct reading frame. These indels can be made using the CRISPR/Cas9 system, which is the aim of this device.
It is important to notice that this device is modular. To insert the region of the gene of interest, this region must have the overhang 5'-AATG-3' and 5'-TTCG-3'
This part was obtained using GoldenBraid assembly. The Ga20ox consense sequence was obtained with oligonucleotide synthesis.
It is a CDS of a bxb1 reporter. It is composed by a terminator T35s flanked by an attB recognition site and an attP recognition site
TALE that induces the BS3 promoter. The part is compatible with the GoldenGate MoClo Assembly Standard (free from BsaI and BpiI recognition sequences) Codon-optimised for expression in dicotyledenous plants
Domestication for GoldenGate by removal of internal BsaI and BpiI recognition sequences. Codon-optimised for expression in dicotyledenous plants
From mycobacterium phage, it is a CDS of bxb1 integrase from serine recombinase superfamilie. It is composed by two exons and an intron from Ricinus Communis which increases integrase effciency.
Half of the SpCas9 protein fused to DnaE C-intein. The active full-lenght spCas9 can be recovered by using the other half of the protein, N-split Cas9 + DnaE N-intein. Inteins are used to fuse both parts of the protein, as they interact and splice, leaving the active protein.
As the part needs to be inserted in a viral vector, the overhangs have to be designed accordingly to the viral vector that is going to be used. The synthesis of C-split Cas9 could not be made in one gBlock, as it is too big. Hence, the half protein had to be splitted in two other parts for its synthesis.
The protein domain is synthesized with the intein.
The part consists of a T35S terminator flanked by BxbI recognition sites. When the integrase is functionally expressed, it makes double strand breaks in the recognition sites so that the terminator is removed.
This part was designed and sent for synthesis. It is plant codon optimized
The reporter was specifically designed for its use in plants
35s + TFL consense + RSIAT-Luciferase + Tnos
Modul of the gRNA testing system created by Valencia UPV 2016 team. This system aims to test the functionality and efficiency of a chosen gRNA to target a particular gene. This system is modular, as the gene inserted can be chosen by the user, only taking into account the required overhangs (5'-AATG-3' and 5'-TTCG-3'). It includes a promoter 35s (BBa_K1537015), 20 nucleotides of the TFL consensus gene of Clemenules (clementine orange), the reporter luciferase with RSIAT linker and the terminator Tnos. Luciferase is out of its reading frame due to a nucleotide added upstream to the linker, and the initial ATG has been removed. This means that when the luciferase is translated, it will not be functional. To make it functional, it is necessary to make indels in the TFL fragment that put the luciferase in the correct reading frame. These indels can be made using the CRISPR/Cas9 system, which is the aim of this device.
It is important to notice that this device is modular. To insert the region of the gene of interest, this region must have the overhang 5'-AATG-3' and 5'-TTCG-3'.
This part was obtained using GoldenBraid assembly. The TFL consense sequence was obtained with oligonucleotide synthesis.
35s + TFL consense + RSIAT-TEV-Luciferase + Tnos
Modul of the gRNA testing system created by Valencia UPV 2016 team. This system aims to test the functionality and efficiency of a chosen gRNA to target a particular gene. This system is modular, as the gene inserted can be chosen by the user, only taking into account the required overhangs (5'-AATG-3' and 5'-TTCG-3'). It includes a promoter 35s (BBa_K1537015), 20 nucleotides of the TFL consensus gene of Clemenules (clementine orange), the reporter luciferase with RSIAT+TEV linker and the terminator Tnos. Luciferase is out of its reading frame due to a nucleotide added upstream to the linker, and the initial ATG has been removed. This means that when the luciferase is translated, it will not be functional. To make it functional, it is necessary to make indels in the TFL fragment that put the luciferase in the correct reading frame. These indels can be made using the CRISPR/Cas9 system, which is the aim of this device.
This biosafety device for plants consists on two submodules: a male sterility submodule and an identity preservation submodule. The male sterility submodule consist on a barnase specifically expressed in anthers under the regulation of the TA29 tapetum-specific promoter. As result, pollen from these plants is not fertile. The identity preservation submodule expresses the blue chromoprotein AmilCP, so that plants can be visually differentiated from non-transgenic plants.
The assembly was performed by GoldenBraid 2.0 assembly. The barnase was amplified by PCR from [http://parts.igem.org/Part:BBa_J72173 Part:BBa_J72173] and a mutation in T105P was introduced.
TA29 promoter [http://parts.igem.org/Part:BBa_K1554000 Part:BBa_K1554000] Barnase [http://parts.igem.org/Part:BBa_J72173 Part:BBa_J72173] AmilGFP [Waiting for Norwich team part name in registry] P35s [http://parts.igem.org/Part:BBa_K1537015 Part:BBa_K1537015]
Diarrheal diseases caused by Vibrio cholera and enterotoxigenic Escherichia coli (ETEC) are worldwide health problems that might be prevented with vaccines based on edible plants expressing the B subunit from either the cholera toxin (CTB) or the E. coli heat labile toxin (LTB). The E. coli heat-labile enterotoxin (LT) is the major ETEC pathogenic factor. LT subunit B (LTB) is of special interest because its immunogenicity, adjuvant activity and lack of toxicity make it a viable antigen to vaccinate against ETEC diarrhea.
The protein consists of the E. coli enterotoxin beta subunit. It is codon optimized for plants and contains a C-terminus 6X Hys tag in order to detected by anti-Hys antibodies.
The LTB protein was designed based on a publication that showed that it can be expressed in Nicotiana Benthamiana plants.
35S:PhiC31:T35S - 35S:ReporterPhiC31::GFP:T35S
Composite composed of the recombinase Bxb1 optimize for Nicothiana Benthamiana and a reporter to prove its action. The reporter composite consist in a terminator flankeated by the recognition sequence of Bxb1 between the promoter and the CDS. Only if the recombinase is present, the GFP will be observed.
Transcriptional units assembled with GoldenBraid
Acyltransferase Candidate 1 -Fused to GFP -Expression Construct
Acylates starch in plants with a GFP tag for localisation
From Pectinidae sp. It is a CDS of green fluorescent protein Dronpa.
Pectinidae sp. sequence found in the following article Lummer, M., Humpert, F., Steuwe, C., Caesar, K., Sch??ttpelz, M., Sauer, M. and Staiger, D. (2011), Reversible Photoswitchable DRONPA-s Monitors Nucleocytoplasmic Transport of an RNA-Binding Protein in Transgenic Plants. Traffic, 12: 693???702. doi: 10.1111/j.1600-0854.2011.01180.x
BS3 is a plant specific promoter which is induced by the TALE AvrBS3. This part is compatible with the GoldenGate MoClo Assembly Standard (free from BsaI and BpiI recognition sequences).
Domestication for GoldenGate (Removal of internal BsaI and BpiI recognition sequences).
This biosafety device for plants consists on two submodules: a male sterility submodule and an identity preservation submodule. The male sterility submodule consist on a barnase specifically expressed in anthers under the regulation of the TA29 tapetum-specific promoter. As result, pollen from these plants is not fertile. The identity preservation submodule expresses the yellow chromoprotein AmilGFP, so that plants can be visually differentiated from non-transgenic plants.
The assembly was performed by GoldenBraid assembly. The barnase was ampliified by PCR from [http://parts.igem.org/Part:BBa_J72173 Part:BBa_J72173] and a mutation in T105P was introduced.
35s + Ga20ox consense + RSIAT-TEV-Luciferase + Tnos
Modul of the gRNA testing system created by Valencia UPV 2016 team. This system aims to test the functionality and efficiency of a chosen gRNA to target a particular gene. This system is modular, as the gene inserted can be chosen by the user, only taking into account the required overhangs (5'-AATG-3' and 5'-TTCG-3'). It includes a promoter 35s (BBa_K1537015), 20 nucleotides of the Ga20ox consensus gene of Oryza sativa (rice), the reporter luciferase with RSIAT+TEV linker and the terminator Tnos. Luciferase is out of its reading frame due to a nucleotide added upstream to the linker, and the initial ATG has been removed. This means that when the luciferase is translated, it will not be functional. To make it functional, it is necessary to make indels in the Ga20ox fragment that put the luciferase in the correct reading frame. These indels can be made using the CRISPR/Cas9 system, which is the aim of this device.
Acyltransferase Candidate 3 Expression Construct
35S is a plant specific promoter obtained from Cauliflower Mosaic Virus. The part is intended for use as a constitutive promoter for gene expression experiments. The part is compatible with GoldenGate MoClo Assembly Standard (free from BsaI and BpiI recognition sequences).
35s + Ga20ox consense + AEK-Luciferase + Tnos
Modul of the gRNA testing system created by Valencia UPV 2016 team. This system aims to test the functionality and efficiency of a chosen gRNA to target a particular gene. This system is modular, as the gene inserted can be chosen by the user, only taking into account the required overhangs (5'-AATG-3' and 5'-TTCG-3'). It includes a promoter 35s (BBa_K1537015), 20 nucleotides of the Ga20ox consensus gene of Oryza sativa (rice), the reporter luciferase with AEK linker and the terminator Tnos. Luciferase is out of its reading frame due to a nucleotide added upstream to the linker, and the initial ATG has been removed. This means that when the luciferase is translated, it will not be functional. To make it functional, it is necessary to make indels in the Ga20ox fragment that put the luciferase in the correct reading frame. These indels can be made using the CRISPR/Cas9 system, which is the aim of this device.
GoldenGate compatible Green Flourescent Protein (GFP)
A variation of the part BBa_E0040 that has been made compatible for Golden Gate by removal of internal BsaI and BpiI recognition sequences. Fluoresces under UV light when the gene is expressed.
Domestication for GoldenGate by removal of internal BsaI and BpiI recognition sequences.
35S:BxbI:T35S - 35S:ReporterBxBI::GFP:T35S
P35s: BBa_K1537015 Bxb1: BBa_K1742005 Bxb1reporter: BBa_K1742006 GFP: Golden braid 2.0 collection T35s: Golden braid 2.0 collection
translation initiation optimized sequence for dicot
35s + TFL consense + AEK-Luciferase + Tnos
Modul of the gRNA testing system created by Valencia UPV 2016 team. This system aims to test the functionality and efficiency of a chosen gRNA to target a particular gene. This system is modular, as the gene inserted can be chosen by the user, only taking into account the required overhangs (5'-AATG-3' and 5'-TTCG-3'). It includes a promoter 35s (BBa_K1537015), 20 nucleotides of the TFL consensus gene of Clemenules (clementine orange), the reporter luciferase with AEK linker and the terminator Tnos. Luciferase is out of its reading frame due to a nucleotide added upstream to the linker, and the initial ATG has been removed. This means that when the luciferase is translated, it will not be functional. To make it functional, it is necessary to make indels in the TFL fragment that put the luciferase in the correct reading frame. These indels can be made using the CRISPR/Cas9 system, which is the aim of this device.
35S:LacIBDBD-DronpaK:T35S - 35S:DronpaN-VP16:T35S
Translational unit composed by the promoter 35S, the T35s terminator, and the CDS Dronpa(145K) and Dronpa(145N). The CDS works as a toggle switch activated with green light and deactivated with blue light. Dronpa(145K) changes its conformation with light, being able to dimerize with Dronpa(145N), acting as a transcription factor.
Dronpa(145K) is synthetized. Dronpa(145N) was obtained by PCR, changing 145K by 145N.
35s + Ga20ox consense + RSIAT-Luciferase + Tnos
Modul of the gRNA testing system created by Valencia UPV 2016 team. This system aims to test the functionality and efficiency of a chosen gRNA to target a particular gene. This system is modular, as the gene inserted can be chosen by the user, only taking into account the required overhangs (5'-AATG-3' and 5'-TTCG-3'). It includes a promoter 35s with a 5' region (BBa_K2017002), 20 nucleotides of the Ga20ox consensus gene of Oryza sativa (rice), the reporter luciferase with RSIAT linker and the terminator Tnos. Luciferase is out of its reading frame due to a nucleotide added upstream to the linker, and the initial ATG has been removed. This means that when the luciferase is translated, it will not be functional. To make it functional, it is necessary to make indels in the Ga20ox fragment that put the luciferase in the correct reading frame. These indels can be made using the CRISPR/Cas9 system, which is the aim of this device.
Acyltransferase Candidate 4 -Fused to GFP -Expression Construct
Acylates starch with a GFP tag for visualisation of localisation
A CDS of Dronpa protein with a mutation in the betha-strand 7 in Lys145 for Asn.
35S:Gal4BD-DronpaK:T35S - 35S:DronpaN-VP16:T35S
Translational unit composed by the promoter 35S, the T35s terminator, the CDS Dronpa(145K), Dronpa(145N), the binding domain Gal4 and VP16. The CDS works as a toggle switch activated with green light and deactivated with blue light. Dronpa(145K) changes its conformation with light, being able to dimerize with Dronpa(145N), acting as a transcription factor.
Half of the SpCas9 protein fused to DnaE C-intein. The active full-lenght spCas9 can be recovered by using the other half of the protein, C-split Cas9 + DnaE C-intein. Inteins are used to fuse both parts of the protein, as they interact and splice, leaving the active protein.
As the part needs to be inserted in a viral vector, the overhangs have to be designed accordingly to the viral vector that is going to be used. The synthesis of N-split Cas9 could not be made in one gBlock, as it is too big. Hence, the half protein had to be splitted in two other parts for its synthesis.
The protein domain is synthesized with the intein
35s:5' + TFL consense + SAGTI-Luciferase + Tnos
Modul of the gRNA testing system created by Valencia UPV 2016 team. This system aims to test the functionality and efficiency of a chosen gRNA to target a particular gene. This system is modular, as the gene inserted can be chosen by the user, only taking into account the required overhangs (5'-AATG-3' and 5'-TTCG-3'). It includes a promoter 35s with a 5' region (BBa_K2017002), 20 nucleotides of the TFL consensus gene of Clemenules (clementine orange), the reporter luciferase with SAGTI linker and the terminator Tnos. Luciferase is out of its reading frame due to a nucleotide added upstream to the linker, and the initial ATG has been removed. This means that when the luciferase is translated, it will not be functional. To make it functional, it is necessary to make indels in the TFL fragment that put the luciferase in the correct reading frame. These indels can be made using the CRISPR/Cas9 system, which is the aim of this device.
Plants and bacteria contain a variety of photoreceptors that activate intracellular signalling in response ti light. Among these, the most extensively studied are LOV (light/oxygen/voltage) domains. On blue light (peak 450nm) aabsorption, the FMN cofactor in the LOV domain forms a covalent bond with a cysteine residue. In the LOV2 domain of plant phototropin, the FMN-cysteine bond formation leads to partial unfolding of the C-terminal alpha-helix from the rest of the LOV2 domain. Strickland et al. created LOV2 fusions capable of light-mediated heterodimerization by fusing the LOV2 domain to peptides that bind to PDZ domains.
The LOv2 domain was fused to a small peptide epitope to the C-terminus of the J-alpha helix (-KAVDTWV). Depending on this peptide, the engineered variants of the Erbin PDZ domains would vary in interaction affinity.
The AsLOV2 fused to a peptide epitope was designed according to the second LOV domain of Avena sativa phototropin (UniprotKB 049003)
Acyltransferase Candidate 2 -Fused to GFP -Expression Construct
Acylates starch with acetyltransferase RV3030 with a GFP tag for visualization of localisation.
This is synthetic a 5'UTR, omega (Tobacco Mosaic Virus. The chloroplast transit peptide provides subcellular localisation to the chloroplast.
It is a CDS of PhiC31 integrase from Streptomyces phage. It has a nuclear location sequence in the N-terminus.
It has a nuclear location sequence (NLS) and its codons are optimized for plants.
Acyltransferase Candidate 4 Expression Construct
Acyltransferase Candidate 2 Expression Construct
PDF1.2 is a plant specific promoter and 5??? untranslated region which is induced by methyl jasmonate. The part is compatible with the GoldenGate MoClo Assembly Standard as it is free from BsaI and BpiI recognition sequences.