constitutive expression of CBDclos fused to BclA with 6xHis tag
CBDclos fused to BclA with 6xHis tag is one of a series of surface expressing fusion proteins that make up biodevice that aims to be therapeutic solution against norovirus infections. This protein in particular is a cellulose binding protein(CBD) fused to surface expression anchoring domain(BclA), connected by a 6xHis tag to be easily identified by Western blotting. For more information, please visit our Wiki page.
The stop codon for this part is not in the coding region, but in the suffix region.
The BclA surface expression component derives from Bacillus anthracis(34F2), it is a hair-like protein that covers B. anthracis??? spores. We used parts BBa_K1933000 from iGEMKyoto 2016 for construction. We outsourced chemical synthesis of this part for construction. CBDclos derives from Clostridium cellulovorans, and is part of a multi-unit cellulosomes. We used parts BBa_K1321341 from iGEM Imperial 2014 for construction.
CBDcex fused to phytochelatin (PC) EC20 with LacI promoter
Phytochelatin (PC) EC20 fused to CBDclos with T7 promoter
The sequence codes for cellulose binding domain cbh2 in prokaryotic cells.
Restriction sites Xho I and Bamh I are added.
Re-entry of BBa_K863111, Cellulose Binding Domain CBDclos in rfc25
This part is a copy of BBa_K863111 (Cellulose binding domain of C. cellulovorans cellulose binding protein gene (Freiburg-Standard)) and was created only as a registry entry, removing the atg-NgoMIV parts at the front and the AgeI-taa basepairs from the sequence so that the registry does not flag it as rfc25 incompatible, and it enables us to enter fusion proteins with this part as automatic composite parts in the method as described by: http://2009.igem.org/Team:Freiburg_bioware/general.
This is a direct copy of BBa_K863111 with slight modifications to the beginning and end of the sequence for the purposes of allowing composite parts to be added correctly using this part.
Phytochelatin (PC) EC20 fused to CBDclos
Coding region for cellulose binding domain. When fused to a protein, protein attaches to cellulose
Sequence was optimized for Escherichia coli and Arabidopsis thaliana codon usage.
Domain derived from cellulose binding protein from Chlostridium cellulovorans(WP_013291799).
NiBP fused to dCBD with linker in RFC 25
constitutive expression of CBDcex fused to BclA with 6xHis tag
CBDcex fused to BclA with 6xHis tag is one of a series of surface expressing fusion proteins that make up biodevice that aims to be therapeutic solution against norovirus infections. This protein in particular is a cellulose binding protein(CBD) fused to surface expression anchoring domain(BclA), connected by a 6xHis tag to be easily identified by Westernblotting. For more information, please visit our Wiki page.
Promoter and RBS:BBa_K1933004 The BclA surface expression component derives from Bacillus anthracis(34F2), it is a hair-like protein that covers B. anthracis??? spores. We used parts BBa_K1933000 from iGEMKyoto 2016 for construction. We outsourced chemical synthesis of this part for construction. CBDcex derives from Cellulomonas fimi, and is part of an exoglucanase secreted by this organism. We used parts BBa_K1321342 from iGEM Imperial 2014 for construction.
SmtA metallothionein fused to linker-dCBD
Phytochelatin (PC) EC20 fused to CBDcex with LacI promoter
Cellulose binding Domain of C. Fimi Exoglucanase with T7, RBS, GS-Linker (Freiburg-Standard)
Cellulose binding domain of the [http://www.ncbi.nlm.nih.gov/nucleotide/327179207?report=genbank&log$=nucltop&blast_rank=3&RID=152ZCN0E01N Cellulomonas fimi ATCC 484 exoglucanase gene] in Freiburg-standard with T7, RBS, GS-Linker (Freiburg-Standard)
You can find additional info to the Cellulose binding domain here: [http://partsregistry.org/Part:BBa_K863101:Design CBDcex_Freiburg]
CBDcenA with linker fused to NiBP in Freiburg format (RFC 25)
CBD2 is EnLinker-CBH2-EnLinker-CBH1 from T.reesei. TBC
Phytochelatin (PC) EC20 fused with CBDclos with LacI promoter
constitutive expression of CBDcex fused to INPNC with 6xHis tag
CBDcex fused to INPNC with 6xHis tag is one of a series of surface expressing fusion proteins that make up biodevice that aims to be therapeutic solution against norovirus infections. This protein in particular is a cellulose binding protein(CBDcex) fused to surface expression anchoring domain(INPNC), connected by a poly-histidine tag to be easily identified by Westernblotting. For more information, please visit our Wiki page.
Promoter and RBS: The INPNC surface expression component derives from Pseudomonas Syringae. We used parts BBa_K811005 from iGEM12_Penn. CBDcex derives from Cellulomonas fimi, and is part of an exoglucanase secreted by this organism. We used parts BBa_K1321342 from iGEM Imperial 2014 for construction.
Double CBD (dCBD) with N-terminal linker
This CBD is from the Cellulosomal -scaffolding protein A (cipA) of Clostridium thermocellum including the endogenous linker sequences at the N and C-terminus (UniProt ID Q06851 link http://www.uniprot.org/uniprot/Q06851). It is in RFC25 format to allow for easy use in protein fusions. The CBD is part of the CBM3 family (link to cazy http://www.cazy.org/CBM3.html). This CBD has been used in many application: in fusions with cell adhesion peptides to enhance the properties of cellulose as a cell-growth matrix (Andrade et al 2010a, Andrade et al 2010b), fused to enzymes to remove contaminants from water (Kauffmann et al 2000) and fused to an antimicrobial peptide (Ramos, Domingues & Gama 2010). At present the cloning for these constructs is still in progress to correct an illegal EcoRI site which was identified in the parts with this CBD. This can be achieved with a silent mutation via site-directed mutagenesis and we aim to send these parts to the registry once this is complete.
The DNA sequence from the relevant portion of http://www.ncbi.nlm.nih.gov/nuccore/144777 was codon optimised for ''E.coli''. RFC[25] prefix and suffix were appended to the sequence with an additional 4 basepairs (gatc) at the beginning and end to allow space for the restriction enzymes to bind to the EcoRI and PstI sites at the ends of the sequence for cloning purposes.
The designed construct was ordered from as a GeneArt?? String (Invitrogen??? Life Technologies).
Cellulose binding Domain of C. cellulovorans cellulose binding protein with Reporter GFP
Cellulose binding Domain from Clostridium cellulovorans [http://www.ncbi.nlm.nih.gov/nuccore/M73817 cellulose binding protein gene] with T7, RBS linked with two amino acids (GS) to a GFP with His-tag.
CBDclos fused to a his-tagged GFP in a Freiburg-manner
Cloning-vector and [http://partsregistry.org/Part:BBa_K863121 BBa_K863121]
Cellulose binding Domain from C. Fimi with const. Promotor and GFP
Cellulose binding Domain from Cellulomonas Fimi with const. Promotor J61101 and Reporter GFP
CBD of C. cellulovorans cellulose binding protein gene with const. Promoter (J23100+J61101) and GFP
Cellulose binding Domain of Clostridium cellulovorans [http://www.ncbi.nlm.nih.gov/nuccore/M73817 cellulose binding protein gene (cbp A)] with a constitutive promoter and RBS (<partinfo>J61101</partinfo>) and a fused-GFP protein with His-tag.
Was made to switch from a T7-promoter to a constitutive promoter
Cellulose binding domain form a cloning-vector. <partinfo>J61101</partinfo> and [http://partsregistry.org/Part:BBa_I13522 GFP] from the partsregistry.
CBDclos fused to Phytochelatin (PC) EC20
Cellulose binding domain of C. cellulovorans cellulose binding protein gene (Freiburg-Standard)
Cellulose binding Domain of Clostridium cellulovorans cellulose binding protein gene (cbp A) http://www.ncbi.nlm.nih.gov/nuccore/M73817
The sequence starts with the Freiburg-prefix (ATG+NgoMIV-restriction-site) followed by 12 Bases (4 AS) of the cellulose binding protein gene in front of the CBD; then 12 bases (4 amino acids) of the cellulose binding protein gene behind the CBD; after that the sequence continues with a short GS-Linker and ends with the Freiburg-suffix (AgeI-restirction-site+TAA).
Phytochelatin (PC) EC20 + Linker-CBDcipA-Linker
CBDclos fused to phytochelatin (PC) EC20 with LacI promoter
Cellulose binding domains with streptavidin domain generator
Different sequences for cellulose-binding domains to prevent homologous recombination. Removed cut sites for RFC-10 compatibility.
Cellulose-binding(clostridium cellulovorans)
Cellulose binding Domain from Clostridium cellulovorans cellulose binding protein
Cellulose binding Domain from Clostridium cellulovorans [http://www.ncbi.nlm.nih.gov/nuccore/M73817 cellulose binding protein gene] in Freiburg-Standard.
Added Freiburg-pre- and suffix for in frame assembly and a two amino acids (GS)-Linker.
Phytochelatin (PC) EC20 fused to linker-dCBD with LacI
constitutive expression of CBDclos fused to INPNC with 6xHis tag
CBDclos fused to INPNC with 6xHis tag is one of a series of surface expressing fusion proteins that make up biodevice that aims to be therapeutic solution against norovirus infections. This protein in particular is a cellulose binding protein(CBDclos) fused to surface expression anchoring domain(INPNC), connected by a 6xHis tag to be easily identified by Western blotting. For more information, please visit our Wiki page.
Promoter and RBS: iGEM16_Kyoto The INPNC surface expression component derives from Pseudomonas Syringae. We used parts BBa_K811005 from iGEM12_Penn. CBDclos derives from Clostridium cellulovorans, and is part of a multi-unit cellulosomes. We used parts BBa_K1321341 from iGEM Imperial 2014 for construction.
Cellulose binding domain of C. cellulovorans with T7, RBS, GS-Linker (Freiburg-Standard)
Cellulose binding domain of the [http://www.ncbi.nlm.nih.gov/nucleotide/327179207?report=genbank&log$=nucltop&blast_rank=3&RID=152ZCN0E01N Cellulomonas fimi ATCC 484 exoglucanase gene] in Freiburg-standard
Added the regulatory unit BBa_K525998 (T7+RBS) in front of the CBD and a short GS-Linker behind the domain and used Freiburg-Standard to be able to fuse proteins to the domain.
CBDcenA-linker fused to Phytochelatin (PC) EC20
CBDclos fused to Phytochelatin (PC) EC20 with T7 promoter
Cellulose binding Domain from Cellulomonas Fimi with Reporter GFP
Cellulose binding Domain of C. Fimi Exoglucanase gene with T7, RBS linked with two amino acids (GS) to a GFP (Freiburg-Standard)
CBDcex fused to a his-tagged GFP in a Freiburg-manner
CBDcex fused to Phytochelatin (PC) EC20 with T7 promoter
sfGFP fused to dCBD with linker in RFC 25
Linker-CBDcipA-Linker + Phytochelatin (PC) EC20
Synthetic phytochelatin EC20 (metal binding peptide) fused C-terminally to CBDcipA (a cellulose-binding domain), which contains an endogenous N and C-terminal linker sequence. At present the site-directed mutagenesis for this construct is in progress to correct an illegal EcoRI site which was identified in the CBDcipA. This construct is part of a library of fusions with cellulose binding domains which we designed to bind to cellulose and enable capture of heavy metals ([http://2014.igem.org/Team:Imperial/Functionalisation project page]). Other fusion parts with this metal binding protein can be seen in the table below: Note that the start and stop codon, plus 6 bp either side of the sequence, are included the RFC25 prefix and suffix which is not shown.
The linker sequences mean fusions should be fine either N or N-terminally to the CBDcipA
Please see part pages for the composite parts
Cellulose binding Domain of Cellulomonas Fimi Exoglucanse (Freiburg-Standard)
Cellulose binding domain of the Cellulomonas fimi ATCC 484 exoglucanase gene
The sequence starts with the Freiburg-prefix (ATG+NgoMIV-restrictionsite) followed by 12 Bases (4 AS) of the exoglucanase gene in front of the CBD. Then 9 bases of the exoglucanase gene behind the CBD. After that the sequence continues with a short GS-Linker.
Cloning-Vector with a CBDcex-Barnase fusion-protein
Re-entry of BBa_K863101, Cellulose Binding Domain CBDcex in rfc25
This part is a copy of BBa_K863101 (Cellulose binding Domain of Cellulomonas Fimi Exoglucanse (Freiburg-Standard)) and was created only as a registry entry, removing the atg-NgoMIV parts at the front and the AgeI-taa basepairs from the sequence so that the registry does not flag it as rfc25 incompatible, and it enables us to enter fusion proteins with this part as automatic composite parts in the method as described by: http://2009.igem.org/Team:Freiburg_bioware/general.
This is a direct copy of BBa_K863101 with slight modifications to the beginning and end of the sequence for the purposes of allowing composite parts to be added correctly using this part.
Phytochelatin (PC) EC20 fused to linker-dCBD with T7 promoter