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.
TEV protease (N-terminal half) - entered sequence may be wrong
N-terminal half of TEV (Tobacco Etch Virus) protease. TEV protease is a highly site-specific protease that is found in the Tobacco Etch Virus (TEV). One of the main uses of this protease is for removing affinity tags from purified proteins. But scientists developed assay for detecting transmembrane protein interactions on its base. TEV protease is splitted into halves (N-terminal and C-terminal). These halves are inactive but assembles and regain activity if they are fused with interacting transmembrane proteins. For detecting TEV protease activity another fusion protein is constructed which is composed of transmembrane region, specific TEV protease cleavage site and kind of transcription factor. When both halves of TEV protease assembles and therefore split protease regains activity it cleaves third protein and transcription factor is released from membrane. Transcription factor is later translocated to the nucleus where induces transcription of promoter with reporter gene. This is N-terminal half of this protease.
Nicola Freyer, Lea Steinbeck, Svenja Meyer, Alexander Deitert
This gene can be used to express subtilisin E in Escherichia coli. Subtilisin E is an alkaline serine protease which non-specifically deigests proteins. To use this part a suitable leader sequence has to be placed in front as the sequence of this BioBrick does not contain a start codon.
The sequence from B. subtilis was codon optimized for E. coli with the DNA and protein sequence analysis software "Geneious" and additionally with the "Codon Optimization Tool" from IDT.
The sequence wasoriginally obtained from a wildtype Bacillus subtilis and was ordered from IDT.
Pepsinogen from Homo sapiens optimized for E. coli
Pepsinogen is the precursor protein for the enzyme pepsin, one of the primary digestive enzymes located in the human stomach. Pepsinogen activates into pepsin in the presence of HCl and other pepsinogen molecules. It is a protease, being used in the human digestive system to digest proteins. This sequence has been codon optimized for expression in E. coli.
Codon optimized for expression in E. coli using Gene Designer from DNA 2.0.
Enteropeptidase from Homo sapiens optimized for E. coli
Enteropeptidase is an enzyme secreted in the small intestine necessary for digestion in humans. It is necessary for trypsin activation and acts as a serine protease.
mutated expression system for subtilisin E in E. coli (Y77W)
This composite part consists of the promoter BBa_R0010, the ribosome binding site BBa_B0034, the newly created BioBrick part BBa_K2020001 and the terminator BBa_B0010. BioBrick BBa_K2020001 is a composite part itself and includes the secretion tag pelB (BBa_J32015) and a subtilisin E gene optimized for ''Escherichia coli'' codon usage (BBa_K2020000). Once introduced into ''E. coli'', this BioBrick is able to produce an inactive version of subtilisin E and simultaneously secret the enzyme into the periplasm of the cell. Subtilisin E is an alkaline serine protease which non-specifically digests proteins. By performing a site-directed mutagenesis, tyrosine77 in the propeptide of the enzyme was exchanged against tryptophan. Therefore, the enzyme loses its proteolytic activity. With this composite part, iGEM Aachen 2016 was able to prove that tyrosine77 in the propeptide of subtilisin E is essential for the activity of the enzyme.
The sequence of the subtilisin E gene from ''Bacillus subtilis'' was codon optimized for ''E. coli'' with the DNA and protein sequence analysis software "Geneious" and additionally with the "Codon Optimization Tool" from IDT. The sequence was partly ordered from IDT (BBa_K2020001 + BBa_B0010) and then cloned into BBa_J04500, a protein expression backbone which already carries the LacI promoter BBa_R0010 and the ribosome binding site BBa_B0034. Afterwards, a mutation in the active site of the enzyme was introduced by performing a site-directed mutagenesis. The codon TAT of tyrosine77 was substituted with TGG which codes for tryptophan.
The sequences of the BioBrick parts were obtained through the Registry. {| class="wikitable" style="text-align:center" |- ! part !! colspan="2"| BioBrick No. |- | LacI promoter || colspan="2"| BBa_R0010 |- | RBS || colspan="2"| BBa_B0034 |- | rowspan="2" | CDS || colspan="2"| BBa_K2020001 |- | BBa_J32015 || BBa_K2020000 |- | terminator || colspan="2"| BBa_B0010 |- |} The construct was ordered at IDT.
mutated expression system for subtilisin E in E. coli (S221X)
This composite part consists of the promoter BBa_R0010, the ribosome binding site BBa_B0034, the newly created BioBrick part BBa_K2020001 and the terminator BBa_B0010. BioBrick BBa_K2020001 is a composite part itself and includes the secretion tag pelB (BBa_J32015) and a subtilisin E gene optimized for ''Escherichia coli'' codon usage (BBa_K2020000). Once introduced into ''E. coli'', this BioBrick is able to produce an inactive version of subtilisin E and simultaneously secret the enzyme into the periplasm of the cell. Subtilisin E is an alkaline serine protease which non-specifically digests proteins. By performing a site-directed mutagenesis, serine in the catalytic triade of the enzyme was exchanged against the amber stop codon UAG. Therefore, the expression of the enzyme is interrupted. This composite part was created to integrate a non-canonical amino acid into the sequence of subtilisin E by adding an orthogonal tRNA/aminoacyl-synthetase pair that is capable of incorporating the non-canonical amino acid of choice at the UAG codon.
The sequence of the subtilisin E gene from ''Bacillus subtilis'' was codon optimized for ''E. coli'' with the DNA and protein sequence analysis software "Geneious" and additionally with the "Codon Optimization Tool" from IDT. The sequence was partly ordered from IDT (BBa_K2020001 + BBa_B0010) and then cloned into BBa_J04500, a protein expression backbone which already carries the LacI promoter BBa_R0010 and the ribosome binding site BBa_B0034. Afterwards, a mutation in the active site of the enzyme was introduced by performing a site-directed mutagenesis. The codon AGC of serine221 was substituted with the stop codon TAG.
mutated expression system for subtilisin E in E. coli (S221Y)
This composite part consists of the promoter BBa_R0010, the ribosome binding site BBa_B0034, the newly created BioBrick part BBa_K2020001 and the terminator BBa_B0010. BioBrick BBa_K2020001 is a composite part itself and includes the secretion tag pelB (BBa_J32015) and a subtilisin E gene optimized for ''Escherichia coli'' codon usage (BBa_K2020000). Once introduced into ''E. coli'', this BioBrick is able to produce an inactive version of subtilisin E and simultaneously secret the enzyme into the periplasm of the cell. Subtilisin E is an alkaline serine protease which non-specifically digests proteins. By performing a site-directed mutagenesis, serine in the catalytic triade of the enzyme was exchanged against tyrosine. Therefore, the enzyme looses its proteolytic activity.
The sequence of the subtilisin E gene from ''Bacillus subtilis'' was codon optimized for ''E. coli'' with the DNA and protein sequence analysis software "Geneious" and additionally with the "Codon Optimization Tool" from IDT. The sequence was partly ordered from IDT (BBa_K2020001 + BBa_B0010) and then cloned into BBa_J04500, a protein expression backbone which already carries the LacI promoter BBa_R0010 and the ribosome binding site BBa_B0034. Afterwards, a mutation in the active site of the enzyme was introduced by performing a site-directed mutagenesis. The codon AGC of serine221 was substituted with TAC which codes for tyrosine.
HIV-1 protease is an aspartyl protease that is essential for the life-cycle of HIV and cleaves proteins at specific aminoacid sequence.
The HtlB (ftsH) coding region from E. coli. It can be used to degrade many different proteins.
Protein coding region for the Hepatitis C virus (HCV) NS3-4A protease with N- & C-terminal linkers. Intended for combination with 145N Dronpa (Part BBa_K1680006) as described by Zhou et al. 2012. Codon optimized for Yeast.
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.
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.
subtilisin E gene with leader sequence pelB
This composite part consists of the leader sequence pelB (BBa_J32015) and the subtilisin E gene (BBa_K2020000). It can be used to express subtilisin E in Escherichia coli and simultaneously secret the enzyme into the periplasm of the cell. Subtilisin E is an alkaline serine protease which non-specifically digests proteins.
The sequence of the subtilisin E gene from Bacillus subtilis was codon optimized for E. coli with the DNA and protein sequence analysis software "Geneious" and additionally with the "Codon Optimization Tool" from IDT. Moreover, the leader sequence pelB begins with a start odon and was placed in front of the subtilisin E gene to generate a functional coding sequence that can be expressed in E. coli.
The sequence of the signal peptide pelB was obtained through the Registry (BBa_J32015: http://parts.igem.org/Part:BBa_J32015). Together with the subtilisin E gene (BBa_K2020000: http://parts.igem.org/Part:BBa_K2020000) it was ordered from IDT.
expression system for subtilisin E in E. coli
This composite part consists of the promoter [http://parts.igem.org/Part:BBa_R0010 BBa_R0010], the RBS [http://parts.igem.org/Part:BBa_B0034 BBa_B0034], the newly created BioBrick part [http://parts.igem.org/Part:BBa_K2020001 BBa_K2020001] and the terminator [http://parts.igem.org/Part:BBa_B0010 BBa_B0010]. BioBrick BBa_K2020001 is a composite part itself and includes the secretion tag pelB ([http://parts.igem.org/Part:BBa_J32015 BBaJ32015]) and a subtilisin E gene optimized for E. coli codon usage ([http://parts.igem.org/Part:BBa_K2020000 BBa_K2020000]). Once introduced into Escherichia coli, this BioBrick is able to produce subtilisin E and simultaneously secret the enzyme into the periplasm of the cell. Subtilisin E is an alkaline serine protease which non-specifically digests proteins.
The sequence of the subtilisin E gene from Bacillus subtilis was codon optimized for E. coli with the DNA and protein sequence analysis software "Geneious" and additionally with the "Codon Optimization Tool" from IDT.
The sequences of the BioBrick parts were obtained through the Registry. The construct was ordered at IDT. add links
Tev Protease, single S219V mutation in the internal cleavage site
TEV protease (also called Tobacco Etch Virus nuclear inclusion a endopeptidase) is a highly sequence-specific cysteine protease from Tobacco Etch Virus (TEV).The consensus for these native cut sites is ENLYFQ\S where ???\??? denotes the cleaved peptide bond. One of the main uses of this protein is for removing affinity tags from purified proteins. The reason for the use of TEV protease as a biochemical tool is its high sequence specificity. This specificity allows for the controlled cleavage of proteins when the preference sequence is inserted into flexible loops. It also makes it relatively non-toxic in vivo as the recognized sequence scarcely occurs in proteins.[1]
This part contains an additional BamHI restriction site at beginning of Tev Protease sequence and a XhoI restriction site at the end.
Microcystinase (Mlra) + mating factor alpha tag, for S. cerevisiae
OmpA is used to present HIV aspartyl protease outside the E. Coli membrane so that it can cut other proteins with the specific sequence outside in the media.
Since HIV protease is a dimer, dimerization is a great concern to its function. Function of this protease is tested in BL21.
HIV protease sequence is from former part BBa_I712667. OmpA sequence is from BL21(DE3) e. Coli strain.
mutated expression system for subtilisin E in E. coli (Y77X)
This composite part consists of the promoter BBa_R0010, the ribosome binding site BBa_B0034, the newly created BioBrick part BBa_K2020001 and the terminator BBa_B0010. BioBrick BBa_K2020001 is a composite part itself and includes the secretion tag pelB (BBa_J32015) and a subtilisin E gene optimized for ''Escherichia coli'' codon usage (BBa_K2020000). Once introduced into ''E. coli'', this BioBrick is able to produce an inactive version of subtilisin E and simultaneously secret the enzyme into the periplasm of the cell. Subtilisin E is an alkaline serine protease which non-specifically digests proteins. By performing a site-directed mutagenesis, tyrosine77 in the propeptide of the enzyme was exchanged against the amber stop codon UAG. Therefore, the expression of the enzyme is interrupted. This composite part was created to integrate a non-canonical amino acid into the sequence of the propeptide of subtilisin E by adding an orthogonal tRNA/aminoacyl-synthetase pair that is capable of incorporating the non-canonical amino acid of choice at the UAG codon.
The sequence of the subtilisin E gene from ''Bacillus subtilis'' was codon optimized for ''E. coli'' with the DNA and protein sequence analysis software "Geneious" and additionally with the "Codon Optimization Tool" from IDT. The sequence was partly ordered from IDT (BBa_K2020001 + BBa_B0010) and then cloned into BBa_J04500, a protein expression backbone which already carries the LacI promoter BBa_R0010 and the ribosome binding site BBa_B0034. Afterwards, a mutation in the active site of the enzyme was introduced by performing a site-directed mutagenesis. The codon TAT of tyrosine77 was substituted with the stop codon TAG.
TEV protease with anti-self cleavage mutation S219V, codon optimized for E. coli
This part is a TEV protease in RFC25 that was optimized for expression in E. coli. The part contains the S219V anti-self cleavage mutation.
The part does not contain start or stop codons, because it was designed for RFC25 compability
The part was codon optimized using the genomic amino acid sequence, designed for RFC25 and mutated S219V for anti-self cleavage properties.
This part is synthesized for the purpose of using it whether our TEV protease based tumor detection system works correctly. TEV protease full structure pretends that its N and C terminus parts are fixed together effectively and that it is ready to cleave target sequences. In our project, there are anchor-inducer protein complexes which are planned to be cleaved by TEV protease. (The cleavage sites are used to fix these two proteins of complex.) After this reaction, it is hopefully possible to see the reporter signal which is released free after the cleavege. This signal will indicate that N and C terminuses of TEV protease are assembled by the effect of detection in the system they used. (In our case, it is the physical antibody-antigen binding effect which TEV protease parts are clustered the intracellular regiın of binding domains)
This part will be assembled with pLac promoter, RBS and LacI sequences in order to be regulated for the control in the assays by the addition of IPTG in the media.
C-terminal half of TEV (Tobacco Etch Virus) protease. TEV protease is a highly site-specific protease that is found in the Tobacco Etch Virus (TEV). One of the main uses of this protease is for removing affinity tags from purified proteins. But scientists developed assay for detecting transmembrane protein interactions on its base. TEV protease is splitted into halves (N-terminal and C-terminal). These halves are inactive but assembles and regain activity if they are fused with interacting transmembrane proteins. For detecting TEV protease activity another fusion protein is constructed which is composed of transmembrane region, specific TEV protease cleavage site and kind of transcription factor. When both halves of TEV protease assembles and therefore split protease regains activity it cleaves third protein and transcription factor is released from membrane. Transcription factor is later translocated to the nucleus where induces transcription of promoter with reporter gene. This is C-terminal half of this protease.
Homo sapiens caspase 3, apoptosis-related cysteine peptidase (transcript variant beta).
Includes start and stop codon. The part is cloned into BBa_J52017, a vector with eukaryotic terminator.
Part comes from Origene SC101958 vector (NM_032991).