SmtA is a metallothionein that can bind to heavy metal ions such as Cd(II). This SmtA is cloned from Synechococcus sp. PCC7942 and it has been reported that the cyanobacterial strains that express SmtA can resist medium containing cadmim.
This part was cloned from Synechococcus sp. PCC7942, using primers to add EcoRI, XbaI and SpeI at the ends.
Derived from the TE terminator of T7 bacteriophage between Genes 1.3 and 1.4 V01146 .
Transcription terminator for the E.coli RNA polymerase.
Suggested by Sri Kosuri and Drew Endy as a high efficiency terminator. The 5' end cutoff was placed immediately after the TAA stop codon and the 3' end cutoff was placed just prior to the RBS of Gene 1.4 (before AAGGAG). Use anywhere transcription should be stopped when the gene of interest is upstream of this terminator.
Bacterioferritin (BFR) M52H heme-deletion
The M52H Mutagensis leads to an deletion of the BFR heme residue. As a result, BFR will have a reduced iron release rate and keep it's iron content inside of the protein scaffold.
This is the scar produced by assembly using RFC 25. If you are assembling a composite part using RFC 25, you can insert this part and specify blunt assembly to get the desired sequence.
Phytochelatin (PC) EC20 fused with CBDclos with LacI promoter
constitutive expression of CBDcex fused to INPNC with 6xHis tag
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.
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.
The stop codon for this part is not in the coding region, but in the suffix region.
RBS + GST (glutathione-S-transferase)-CRS5 (metallothionein) + Ter
This part is BBa_K1460001 with the T7 promoter removed.
his part serves as a functional metallothionein for heavy metal binding and conferred resistance to heavy metals when combined with an appropriate promoter. CRS5 gene is the metallothionein that binds to heavy metals and it is fused to GST for stability.
The part contains a KpnI cut site between the biobrick prefix and the RBS that is a result of removing the T7 promoter from part BBa_K1460001.
Phytochelatin (PC) EC20 fused to CBDclos
Coding for the trans-acting regulator SmtB
Metallothioneins(MTs), discovered in horse kidney in 1957 by Margoshes and Vallee are identified as low-molecular weight and sulphydryl rich proteins which bind metal ions in metal-thiolate clusters and whose synthesis increases in response to elevated concentrations of certain metals. In 1988, Olafson and co-workers reported the amino acid sequence of a metal-associated protein isolated from cyanobacterium Synechococcus sp.. With the function of binding metal ions remaining, it only has less than 20% homology in amino acid sequence with known MTs in eukaryotes and thus we call it class II MT or on some occasions MT-like. Owing to the works of Robinson and Huckle, the corresponding locus of Synechococcus PCC7942, smt locus was isolated and analyzed. It includes smtA which encodes the class II MT and a divergently transcribed gene smtB which encodes a trans-acting, autoregulatory repressor that exerts metal ion-inducible negative control over smtA transcription. Between them lies a 100-bp operator-promoter region(smt O-P, BBa_K1509001), which is responsible for the divergent orientation of smtA and smtB.
In 1993, Huckle and co-workers reported the isolation and analysis of the smt locus from Synechococcus sp.. Gene smtB, one of the components of the locus, encodes a transcriptional repressor of gene smtA, with which is required for normal tolerance to Zn2+ and Cd2+. The product of smtB functions by binding to the smt operator-promoter(smtO-P) and dissociating in the presence of Zn2+ due to the classical helix-turn-helix motif similar to many DNA-binding proteins. It can also be genomically rearranged when induced by Cd2+, leading to a deletion within smtB because of a highly iterated palindrome (HIP1), a highly represented octanucleotides(5′-GCGATC-GC-3′) which traverse both borders of the excised element. These two mechanisms both cause the elevated expression of smtA.
According to the mechanisms of smt locus represented above, gene smtB should function with the accompany of smt operator-promoter. Previous cases selected smtA whose product functions by encoding MT-like to sequester metal ions, while our case intended to take advantage of the trigger lies in this locus. By imitating former researches, in which a promoter less lacZ gene was fused upstream of smtA, we respectively splice two selected reporter genes and sequence from smt operator-promoter to smtB, equivalent to replacing smtA with reporter genes, to visualize the effects of metal ions on the locus.
Anderson promoter is that of part BBa_J23102. Terminator is that of part BBa_B1006. NixA gene is naturally found in H. pylori. This part was synthesized by GenScript in the vector pUC57. Part was then transferred into pSB1C3 using standard cloning procedures.
This is a functional coding sequence for the protein NixA, which is a nickel transporter from H. pylori. This part will result in nickel intolerance from higher levels of intracellular nickel.
The Anderson promoter provides strong constitutive expression of NixA in E. coli.
Source organism: Serratia marcescens. Amplified from plasmid pDU1358, kindly donated by Anne O. Summers.
merT is found in the Serratia marcescens plasmid pDU1358 on the other side of the bidirectional promoter opposite of merR. It is 351 base pairs long and is hypothesized that it codes for a mercuric transport protein.
N-terminal domain of BclA protein fused to 6xHis tag
NCBI accession number CAD56880 NCBI accession number CAD56878 NCBI accession number CAD56869 NCBI accession number CAD56870
This part codes N-terminal domain of BclA protein which expressed on the cell surface connected by a 6??His tag to be easily identified by Western blotting.[1] Surface displayed passengers retain enzyme activity even when it is linked to BclA with 6?? His tag.
optimized to expression in E. coli K-12 strain
The OprF gene is from Pseudomonas aeruginosa.
The OprF is a major outer membrane protein of Pseudomonas aeruginosa. This protein functions as a nonspecific porin to allow the passage of small hydrophilic molecules, plays a structural role in maintaining cell shape and outer membrane integrity, and is required for growth under low osmolality. The structure of OprF has been proposed to consist of three domains, the N-terminal forming h-barrel structure, a loop or hinge region, and the C-terminal associated with peptidoglycan. Based on the predicted secondary structure and information found in the literature, we chose Ala196 as potential fusion sites for displaying CBP. CBP is the abbreviation of copper binding peptide made up of seven amino acid.
Based on the predicted secondary structure and information found in the literature, we chose Val(188) as potential fusion sites for displaying CBP. CBP is the abbreviation of copper binding peptide made up of seven amino acid.
Phytochelatin (PC) EC20 + Linker-CBDcipA-Linker
The gene was functionally expressed in Azospirillum sp. B510. It encodes a divalent metal cation transporter. The protein can be used to increase transportation level of divalent metal ions into the cells. Its potential applications include heavy metal bioremediation (by increasing uptake efficiency) and protein expression in cell surface (by fusion with mntH).
RBS (Elowitz 1999) -- defines RBS efficiency
Varies from -6 to +1 region from original sequence to accomodate BioBricks suffix. No secondary structures are formed in the given RBS region. Users should check for secondary structures induced in the RBS by upstream and downstream elements in the +50 to -50 region, as such structures will greatly affect the strength of the RBS. Contact info for this part: Brian Chow
Vinay S Mahajan, Voichita D. Marinescu, Brian Chow, Alexander D Wissner-Gross and Peter Carr IAP, 2003.
The MerB gene codes for the proetin which function is to cleave the organic part of the molecule of methyl-mercury
The ferric uptake repressor of Bacillus subtilis called YqkL is a regulator that when in contact with iron will bind and repress a regulatory sequence (the Fur box).
We sequenced the Fur box. This can be added in front of genes to provide regulation when in contact with iron.
The OprF is a major outer membrane protein of Pseudomonas aeruginosa. This protein functions as a nonspecific porin to allow the passage of small hydrophilic molecules, plays a structural role in maintaining cell shape and outer membrane integrity, and is required for growth under low osmolality.The structure of OprF has been proposed to consist of three domains, the N-terminal forming h-barrel structure, a loop or hinge region, and the C-terminal associated with peptidoglycan.
Based on the predicted secondary structure and information found in the literature, we chose Val(188）as potential fusion sites for displaying CBP.CBP is the abbreviation of copper binding peptide made up of seven amino acid.In order to reduce the effect on the CBP activity，we added (G4S) linker between OprF and CBP .
RBS (B0034) + MerR (mercury-responsive transcription factor)
The coding sequence of MerR comes from Tn, prefixed by an RBS part BBa_B0034 from Partsregistry
This part was designed as a translational unit for MerR expression.
The native RBS of MerR is not strong enough and its intensity can not be predicted as there is no such a corresponding RBS part in Registry. In order to facilitate the use and further characterization of MerR for future team, we prefixed an RBS part BBa_B0034 from Partsregistry.
Mycobacterium tuberculosis. Ordered as codon optimised DNA.
MymT is a small prokaryotic copper metallothein discovered in Mycobacterium tuberculosis. It is believed that the protein may help the bacterium survive copper toxicity, though deleting the gene had no effect on pathogenicity in mice. It can bind up to 7 copper ions but has a preference for 4-6. This version has a C terminal sfGFP with a C terminal hexahistidine tag for purification. The MymT and sfGFP are separated by a small flexible linker. This part has been codon optimised for E. coli.
This protein has a C terminal hexahistidine tag for purification and has been codon optimised for E. coli.
Phytochelatin (PC) EC20 fused to CBDclos with T7 promoter
Sequenced sourced from Professor David Wilson, of the Department of Molecular Biology and Genetics, Cornell University.
MerT is a transporter responsible for the crossing of the inner membrane by mercury. It enhances mercury uptake from the environment.
Standard 23 prefix and suffix was added to the sequence.
Gold-specific binding protein (GolB) is a gold-binding chaperon discovered in S. Typhimurium which has been verified that the display of GolB on E.coli cell surface permitted selective enrichment of gold ions from media containing various metal ions.
This protein was selected from a pIII phage display library based on its ability to nucleate ZnS or CdS nanocrystals.
J140, with a sequence of Ser-Leu-Thr-Pro-Leu-Thr-Thr-Ser-His-Leu-Arg-Ser, has been shown to recognize single-crystal CdS and nucleate quantum dots (Mao, C., et al. "Viral assembly of oriented quantum dot nanowires" PNAS 100:6946, 2003)
We wanted to be able to clone this part into the IPTG-inducible expression vector pET28b, so we engineered in restriction sites that would allow us to do so and keep everything in frame. Therefore the part contains an Nco1 site after the Xba1 site, and a BamH1 site before the Spe1 site.
Cellulose binding Domain from Cellulomonas Fimi with Reporter GFP
Cloning-vector and [http://partsregistry.org/Part:BBa_K863121 BBa_K863121]
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
Domain derived from cellulose binding protein from Chlostridium cellulovorans(WP_013291799).
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.
BmtA (Lead/Zinc-binding metallothionein from Oscillatoria brevis, codon optimized for E. coli)
The sequence used for the BmtA (metallothionein) was codon optimized for E. coli (using the browser-based software, Gene Design). The native sequence comes from Oscillatoria brevis, and was sourced from an entry in the NCBI database by Liu,T., Nakashima,S., Hirose,K., Uemura,Y., Shibasaka,M., Katsuhara,M. and Kasamo,K. (2003).
This is the BmtA coding sequence from Oscillatoria brevis, which encodes lead and zinc binding metallothionein (Liu et al., 2003). The BmtA sequence in this part has been codon optimized for E. coli. This part can be used with a constitutive or inducible promoter to produce metallothionein in projects utilizing the metal-binding protein for sequestration purposes.
When used in E. coli for the sequestration of lead or zinc ions in solution, the part may or may not work better in concert with a genomic ZntA knockout. We plan to characterize this combination in the near future.
NiBP fused to dCBD with linker in RFC 25
Cellulose binding Domain of Cellulomonas Fimi Exoglucanse (Freiburg-Standard)
Cloning-Vector with a CBDcex-Barnase fusion-protein
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.
Sequence is not natural. Basis for the sequence was from the following reference: Bae W, Chen W, Mulchandani A, Mehra RK (2000) Enhanced bioaccumulation of heavy metals by bacterial cells displaying synthetic phytochelatins. Biotechnol Bioeng. 70(5):518-24.
Synthetic Phytochelatin EC(20) rfc25 fusion format
RBS is predicted from pmsp3 promoter from Magnetospirillum magneticum strain AMB-1 genome. smtA sequence is from BioBrick part BBa_K190021 (we modified the illegal PstI site).
The RBS is from pmsp3 promoter, the strongest promoter isolated from Magnetospirillum bacteria. SmtA is a metallithionein isolated from E.coli and it binds covalently to divalent metal cation. It can usually binds 3-4 ions per protein, depending on the ion types. It is often used to increase the accumulation of metal ions intracellularly and potentially improve bacteria's tolerance of metal ions. The SmtA protein and mCherry protein are linked by a fusion protein linker. The purpose of the linkage is to improve the expression/folding of SmtA and for better visualization of successful transformation. The original use is to express SmtA in Magnetospirillum magneticum strain AMB-1.
We researched the optimum linker sequence for fusion protein to facilitate successful fusion. The mCherry protein is expected to improve the folding of SmtA (from E.coli) in AMB-1.
RBS.1 (strong) -- modified from R. Weiss
Strong RBS based on Ron Weiss thesis. Strength is considered relative to <bb_part>BBa_B0031</bb_part>, <bb_part>BBa_B0032</bb_part>, <bb_part>BBa_B0033</bb_part>.
Varies from -6 to +1 region from original sequence to accomodate BioBricks suffix ("orig" in figure 4-14 of Ron Weiss thesis). No secondary structures are formed in the given RBS region. Users should check for secondary structures induced in the RBS by upstream and downstream elements in the +50 to -50 region, as such structures will greatly affect the strength of the RBS. Contact info Brian Chow
pT7 + RBS + GST (glutathione-S-transferase)-CRS5 (metallothionein) + Ter
T7 promoter is part BBa_I712074. RBS is from part BBa_J61101. Glutathione-S-transferase is from S. japonicum. CRS5 is a metallothionein from S. cerevisiae. Terminator is part BBa_B1006. This sequence was synthesized by GenScript in the vector pUC57 and was then transferred using traditional cloning into pSB1C3.
This part serves as a functional metallothionein for heavy metal binding and conferred resistance to heavy metals when combined with a strain expressing the bacteriophage T7 polymerase. CRS5 gene is the metallothionein that binds to heavy metals and it is fused to GST for stability.
Flanking the T7 promoter are two KpnI cut sites with both the T7 promoter and a SacI cut site between them. These cut sites are present for easy removal of the T7 promoter for modulation of parts with other promoters. The SacI cut site can be used as a marker for successful removal of the T7 promoter from the part. When digested with SacI, part BBa_K1460001 in pSB1C3 will yield bands of size 2166bp and 931bp. If the T7 promoter is successfully removed, when digested with SacI the plasmid will yield the linearized length of the pSB1C3+BBa_K1460001
Re-entry of BBa_K863111, Cellulose Binding Domain CBDclos in rfc25
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.
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.
Phytochelatin (PC) EC20 fused to linker-dCBD with LacI
Transcriptional terminator consisting of a 64 bp stem-loop.
constitutive expression of CBDclos fused to BclA with 6xHis tag
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.
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.
RBS(B0034)_MBP(lead metal binding peptide egineered from PbrR)+Terminator(B0015)
This part is combined with a rbs, a mbp which codes for the metal binding peptide and a terminator.
pCopA TAT Csp1 sfGFP with divergent CueR
pCopA TAT Csp1 sfGFP with plasmid constitutively expressed CueR
merB, Organomercurial Lyase from Serratia marcescens
Amplified from pDU1358, kindly donated by Anne O. Summers.
constitutive expression of CBDclos fused to INPNC with 6xHis tag
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.
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.
sfGFP in Freiburg format (RFC 25) to allow for easy use in fusion proteins.
Ptac-NiCoTB, improves nickel and cobalt internalization
This part comprises a strong E. coli promoter with...
MerP, mercuric transport protein periplasmic component
Source organism: Serratia marcescens. Plasmid pDU1358 kindly donated by Anne O. Summers.
merP is in charge of periplasmic transportation of mercury. This protein has two conserved cysteine residues for specific binding with the mercury ion, Hg(II).
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
Double CBD (dCBD) with N-terminal linker
Anderson Promoter + nixA + Ter + pT7 + GST-CRS5 + Ter
This part is a composite of BBa_K1460003 and BBa_K1460001.
This composite part functionally codes for the selective nickel transporter nixA as well as the metallothioniein CRS5 fused to glutathione-S-transferase. This composite part should act to transport nickel ions into the engineered cell where they will be sequestered by the metallothionein allowing for Ni2+ removal from contaminated water.
Expression of GST-CRS5 is controlled by the bacteriophage T7 promoter, so part should be combined with a host organism expressing T7 polymerase (such as E. coli BL21-AI
The source organism for the main protein sequence is Methylosinus trichosporium OB3b. The TAT sequence originate from E. coli multi copper oxidase enzyme CueO. We ordered it as codon optimised DNA from IDT.
Copper Storage protein 1 (Csp1) is a tetrameric copper storage protein found in the periplasm of Methylosinus trichosporium OB3b. We investigated whether this part could act as a copper chelator when expressed in E. coli. We modified the protein by adding a TAT signal peptide from the E. coli enzyme CueO in place of the native TAT sequence and a C terminal his tag in an attempt to direct the protein to the periplasm.
Codon optimised for E. coli. TAT sequence changed to an E. coli TAT sequence.
constitutive expression of CBDcex fused to BclA with 6xHis tag
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.
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.
CBDcenA-linker fused to Phytochelatin (PC) EC20
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.
Strong RBS + merA (mercuric ion reductase)+ terminator (BBa_ B0015)
MerA gene sequence is found in the O26-CRL plasmid from Escherichia coli O26. We also added strong RBS from protein 10 found in phage 17 and the double terminator (BBa_B0015) to ensure efficient termination of transcription and messenger RNA recognition.
The MerA gene is one of the most important gene into bacterial mercury-resistance operon. Transforms mercury Hg2+ in Hg0, which is volatile and moves passively out of the bacteria in gaseous form as shown in Figure above. The reduction of Hg2+ occurs by a NADPH-dependent system.
We analyzed all Mercury-resistance (Mer) operon genes, in these following strains available at NCBI: (1) Escherichia coli 042, (2) Escherichia coli O26 , (3) Escherichia coli ACN001, (4) Escherichia coli 838C-R1, (5) Escherichia coli APEC-O103, (6) Escherichia coli UMNK88, (7) Escherichia coli AR060302, (8) Escherichia coli PMV-1. It is showed that the mer genes from strains (1) - (2) - (3) - (4) - (5) is conserved among them. However strains (6) - (7) and (8) did not show any similarity. Therefore we used sequences from (1) to (5) due the consensus in E.coli.
Double terminator consisting of BBa_B0010 and BBa_B0012
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]
RBS is predicted from pmsp3 promoter from Magnetospirillum magneticum strain AMB-1 genome. MntH is from part BBa_K1505000. mCerulean is from part BBa_K133066.
The RBS is from pmsp3 promoter, the strongest promoter isolated from Magnetospirillum bacteria. mntH was functionally expressed in Azospirillum sp. B510. It encodes a divalent metal cation transporter. The MntH protein and mCerulean protein are linked by a fusion protein linker. The purpose of the linkage is to improve the expression/folding of MntH and for better visualization of successful transformation. The original use is to express MntH in Magnetospirillum magneticum strain AMB-1.
We researched the optimum linker sequence for fusion protein to facilitate successful fusion. The mCerulean protein is expected to improve the folding of MntH in AMB-1.
MymT was cloned from pGB68 (kindly provided by Ben Gold, Cornell University), the gene's origin is the genome of ''M. tuberculosis''
MymT is a methallothionein binding Cu (II)
BioBrick prefix and suffix was added by PCR, also the RBS (B0034) was added by PCR (not in the sequence yet).
SmtA metallothionein in Freiburg format (RFC[25])
SmtA metallothionein in rfc25 format. This part is based on BBa_____ but has been codon optimised for E.coli and is in the rfc25 biobrick format for ease of use as part of protein fusions. This part is available as fusions as follows:
Codon optimised for E.coli. rbs not included in the part as it is for fusions
pCopA with plasmid constitutively expressed CueR (no reporter)
Codon optimised. Designed for biobrick assembly with reporter of choice
CBD of C. cellulovorans cellulose binding protein gene with const. Promoter (J23100+J61101) and GFP
Cellulose binding domain form a cloning-vector. J61101 and [http://partsregistry.org/Part:BBa_I13522 GFP] from the partsregistry.
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 ( J61101 ) and a fused-GFP protein with His-tag.
Was made to switch from a T7-promoter to a constitutive promoter
sfGFP fused to dCBD with linker in RFC 25
Anderson Promoter + merT + merP + Ter + pT7 + GST-CRS5 + Ter
This part is a composite of parts BBa_K1460004 and BBa_K1460001
This composite part functionally codes for the selective mercury transporters merT and merP as well as the metallothioniein CRS5 fused to glutathione-S-transferase. This composite part should act to transport mercury ions into the engineered cell where they will be sequestered by the metallothionein allowing for Hg2+ removal from contaminated water.
Expression of GST-CRS5 is controlled by the bacteriophage T7 promoter, so part should be combined with a host organism expressing T7 polymerase (such as E. coli BL21-AI from Life Technologies).
Phytochelatin (PC) EC20 fused to CBDcex with LacI promoter
Regulation and transport of mercury ions
This construction is based on sequences present in the O26-CRL plasmid found in Escherichia coli O26. We added a reverse tryptophan operon terminator to ensure transcription termination of the merR messenger RNA. However we didn???t add transcription terminator after the merP gene, aiming connect this biobrick with others, enabling various functions related to mercury
We call this part an ???Essential biobrick??? designed to be the key piece of any genetic construction related to mercury, targeting both a biosensor as a bioaccumulative. This biobrick has bidirectional promoter, the regulator MerR with a terminator reverse from the tryptophan operon and a forward MerP and MerT transporters.
If is desirable to accumulate mercury, assemble this biobrick with a good RBS, a coding sequence of a peptide having affinity to metal and a terminator! Its function will be regulated by presence of mercury through MerR regulator. The bacteria will carry the mercury to cytoplasm until it is linked to the peptide having affinity to the metal, making a inactive mercury. Is not it amazing? This is the strategy that we use in our project! The so called ???Essential Biobrick??? was connected to biobrick for bioremediation, bioaccumulation and bio-detection!
A lead-specific binding protein, PbrR, and promoter pbr from the lead resistance operon, pbr, of Cupriavidus metallidurans CH34 have shown highly sensitive and selective whole-cell detection of lead ions.
Cellulose binding Domain from C. Fimi with const. Promotor and GFP
Cellulose binding Domain from Cellulomonas Fimi with const. Promotor J61101 and Reporter GFP
Anderson Promoter + CBP4 + pT7 + GST-CRS5 + Ter
This part is a composite of the parts BBa_K1460005 and BBa_K1460001.
This composite part functionally codes for the putative lead transporter CBP4 as well as the metallothioniein CRS5 fused to glutathione-S-transferase. This composite part should act to transport lead ions into the engineered cell where they will be sequestered by the metallothionein allowing for Pb2+ removal from contaminated water.
pCopA MymT sfGFP with plasmid constitutively expressed CueR
Anderson promoter is that of part BBa_J23102. Terminator is that of part BBa_B1006. merT/merP genes are naturally found in Pseudomonas aeruginosa. This part was synthesized by GenScript in the vector pUC57. The part was then transferred into pSB1C3 using standard cloning procedures.
This is a functional coding sequence for the mercury transport proteins merT/merP from P. aeruginosa. This part will result in mercury intolerance due to greater mercury transport into engineered cells.
The Anderson promoter provides strong constitutive expression of merT/merP in E. coli
SmtA metallothionein fused to linker-dCBD
The Plac insert was PCR'd from the MG1655 strain of E.coli K12.
Inverting regulatory region controlled by LacI (<bb_part>BBa_C0010</bb_part>, <bb_part>BBa_C0011</bb_part>, etc.) The pLac regulatory region is a 243 base-pair sequence with standard BioBrick prefix and suffix sections on its ends. It contains two protein binding sites: CAP, which is generally present in E.coli and is assocciated with cell health and availability of glucose., and LacI, the Lac inhibitor <bb_part>BBa_C0010</bb_part> which binds in an dimerized cooperative manner to inhibit the transcription of the protein that follows. In the presence of lactose or IPTG, an analog of lactose, LacI is unable to correctly bind and inhibit transcription. This allows <bb_part>BBa_R0010</bb_part> to be used as a inverter or as a detector of lactose or IPTG.
LacI binds to this regulator. This part is incompatible with species containing active LacI coding regions. Lactose and IPTG disable the operation of LacI and this regulator. This part is incompatible with environments containing lactose or lactose analogs.
MymT is a small prokaryotic copper metallothein discovered in Mycobacterium tuberculosis. It is believed that the protein may help the bacterium survive copper toxicity, though deleting the gene had no effect on pathogenicity in mice. It can bind up to 7 copper ions but has a preference for 4-6. Our version has a C terminal hexahistidine tag for purification and has been codon optimised for E. coli.
The sequence codes for cellulose binding domain cbh2 in prokaryotic cells.
Restriction sites Xho I and Bamh I are added.
Bacterioferritin from E. coli Nissle 1917 genomic DNA. Amplified by using PCR and cloned into pQE80L
Bacterioferritins are the E. coli cells natural iron storage proteins. These hollow nearly spherical protein shells detoxify the cell by sequestering excessive iron and forming Iron(III)hydroxid-oxide particels. Bacterioferritin is an heam containing bacterial ferritin. Each heme is bound in a pocked formed by the interface between a pair of symmetry-related subunits [1]. However, it was investigated that these heme groups may be involved in the release of iron out of the ferritin iron core by forming an heme-mediated electron transfer to reduce immobilized Fe3+ to more soluble Fe2+. [1] Frolow F, Kalb AJ, Yariv J. Structure of a unique twofold symmetric haem-binding site. Nat Struct Biol. 1994 Jul;1(7):453-60. PubMed PMID: 7664064. [2] Yao H, Wang Y, Lovell S, Kumar R, Ruvinsky AM, Battaile KP, Vakser IA, Rivera M. The structure of the BfrB-Bfd complex reveals protein-protein interactions enabling iron release from bacterioferritin. J Am Chem Soc. 2012 Aug 15;134(32):13470-81. doi: 10.1021/ja305180n. Epub 2012 Aug 1. PubMed PMID: 22812654; PubMed Central PMCID: PMC3428730.
For any metal nanoparticles built inside of BFR it would be beneficary to remove any heme groups in order to prevent unloading of BFR by heme-mediated electron transfer.
merR family transcriptional regulator, regulatory protein for mer operon
merR, located upstream of the rest of the mer genes, serves to regulate the operon. The MerR protein contains a C-terminal effector binding region that recognizes ionic mercury. Upon binding it, the protein???s N-terminal helix-turn-helix DNA binding domain can bind the merR promoter region to induce transcription of the operon. The effector binding region of the protein can vary allowing for great diversity in merR-like promoters that can respond to a variety of heavy metals as well as antibiotics and oxidative stress.
INPNC fused to 6xHis tag without stop codon
INPNC codes for N- and C- terminal domain of Ice-Nucleation Protein (INP) from Pseudomonas syringae , connected by a 6xHis tag to be easily identified by Western blotting[1]. This is used as an anchoring motif for the cell surface display of passengers. This domain does not have signal peptide for anchoring on the cell surface[2]. Surface displayed passengers retain enzyme activity even when it is linked to INPNC with 6xHis tag.
We omitted stop codon from BBa_K1933001 to use it for the registration of composite parts.
Arabidopsis thaliana (Columbia; leave) Pseudomonas Putida KT2240 (synthsized)
This Fusionprotein was obtained by fusion PPMT and ATPCSand connecting both through the Freiburg GS-Linker PPMT Pseudomonas Putida metallothionein ATPCS Arabidopsis thaliana Phytochelatin synthase
Accodording to he paper Park et al the coexpression of both proteins leads into the formation of an inclusionbody like nanoparticle. Which leads to intercelluar synthesis of diverse nanoparticles.
CBDclos fused to phytochelatin (PC) EC20 with LacI promoter
Phytochelatin (PC) EC20 fused to linker-dCBD with T7 promoter
CBDcenA with linker fused to NiBP in Freiburg format (RFC 25)
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.
CP47 His-tagged with Lac Promoter synthesized by IDT DNA Inc.
Inducible High Expression His-Tagged Photosystem II CP47 subunit
Ferric ion-binding protein (FbpA) without a signal peptide
This part encodes a protein capable of binding a ferric ion. The periplasmic signal peptide has been deleted in order to achieve cytoplasmic localisation of the protein.
Deletion of a putative native periplasmic signal peptide
Transcription Factor Binding Site from Cyanobacterial Pcpc560
merA, mercuric reductase from Serratia marcescens
Mercuric reductase catalyzes the reduction of Hg to Hg2+.
Mutagenesis of adenine to guanine at position 1026 to remove EcoRI site.
CBDclos fused to Phytochelatin (PC) EC20 with T7 promoter
Anderson promoter is that of part BBa_J23102. CBP4 is a naturally found coding sequence in Nicotiana tabacum.[1] This part was synthesized by BioBasic and was transferred into pSB1C3 using standard cloning procedures.
This is a functional coding sequence for the putative lead and nickel transport protein CBP4 from Nicotiana tabacum. It has been found that expression of this protein leads to increased nickel tolerance and lead intolerance.
The Anderson promoter will provide strong constitutive expression of the CBP4 gene in E. coli.
iGEM Calgary 2013 from a synthesized codon optimized construct
Human ferritins do loads more iron atoms then bacterial ferritins. Furthermore they are forming metall cores that are less contaminated with phosphates then e.g. bacterial ferritins. Due to this fact, this should result in a purer iron crystal with an improved magnetic moment.
This design was provided by iGEM Calgary and the HuFerritin as a single protein coding biobrick was constructed by iGEM Berlin as this biobrick was not accessible in this version in the registry.
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)
This parts encodes a protein capable of binding ferric ion. The protein includes a signal peptide targeting it to periplasmic space.
CDS7 cadmium sulfate binding peptide coding sequence
This part was synthesized de novo based on the information in the original paper. It was PCR'd out of the synthesized plasmid with RFC 23 overhangs and ligated into pSB1C3 vector.
This sequence codes for a short peptide that's been reported to bind to cadmium sulfate and nucleate quantum dot formation. (C. Mi et al. / Journal of Biotechnology 153 (2011) 125???132) Peptide sequence: N-GDVHHHGRHGAEHADI-C
Original coding sequence in the referenced paper had NcoI and BamHI sites surrounding the coding sequence for cloning into pET28b IPTG inducible vector. For our purposes we eliminated the restriction sites and instead replaced them with standard biobricking prefix and suffix.
The designed construct was ordered from as a GeneArt?? String (Invitrogen??? Life Technologies).
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.
CBD2 is EnLinker-CBH2-EnLinker-CBH1 from T.reesei. TBC
pCopA MymT with divergent expressed CueR
pCopA MymT with plasmid constitutively expressed CueR
Gold binding peptides (GBP) is a kind of peptide which can bind on gold, usually used to immobilize protein on gold surface.
SmtA is a metallothionein that can bind to metal ions such as Cd(II).
This part is put together with Double terminator.
CBDcex fused to Phytochelatin (PC) EC20 with T7 promoter
Nickel Binding Protein (NiBP) Freiburg format (RFC[25])
This part is based on the existing BioBrick BBa_K1151001 by the 2013 Salente Lecce iGEM team.
Nickel Binding Protein in Freiburg format (RFC 25) to allow for easy use in fusion proteins.
OEPCR primers were designed to allow for the replacement of the standard BioBrick prefix and suffix with the Freiburg prefix and suffix.
The gene was cloned from pET29a-SmtA (kindly provided by Kevin Waldron, Newcastle University), it's origin is from the genome of ''Synechococcus PC''
SmtA is a metallothionein that binds Zn (II) ions, it is able to bind 3-4 ions per protein.
The BioBrick prefix and suffix were added by PCR, also the RBS (B0034) was added.
CBDclos fused to Phytochelatin (PC) EC20
We have to optimizes its codons in Arabidopsis Thaliana and removed the restriction sites for EcoR1, Xba1, Spe1 and Pst1.
This sequence is produced by an assembly method.
This sequence is produced by RFC10-assembly of a RFC10-RBS with a RFC25-prefixed part.
The resulting sequence is a combination of the RFC10 RBS-CDS scar (tactag) and 9 nucleotides from the RFC25 prefix (atg gcc ggc).
fMT metallothionein in Freiburg format (RFC[25])
fMT metallothionein in rfc25 format. This part is based on BBa_____. It can be found as fusions in the following parts:
RBS(B0032)+MBP(mercury metal binding peptide engineered from MerR)
This part is a combination of a rbs, a mbp, coding a mercury metal binding peptide egineered from MerR, and a terminator.
Re-entry of BBa_K863101, Cellulose Binding Domain CBDcex in rfc25
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.
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.
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).
Naoki Saito,Daiki Haraguchi,Yoshiharu Otaki,Ryuhei Minei
Linker-CBDcipA-Linker + Phytochelatin (PC) EC20
Please see part pages for the composite parts
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
fMT was cloned from pMAL-MT (kindly provided by Wilfred Chen, University of California), the origin of the gene is the genome of Fucus vesiculosus.
fMT is a metallothionein binding Arsenite (III) and Arsenate(V), used to accumulate As when overexpressed. Binds As (III) stronger than As(V) Reference: Highly Selective and Rapid Arsenic Removal by Metabolically Engineered Escherichia coli Cells Expressing Fucus vesiculosus Metallothionein. Singh et al. App and Env Microbiology, May 2008, p. 2924???2927
Add BioBrick prefix and suffix, add RBS (B0034)
Derived from peptide described in: Pazirandeh et al. Development of Bacterium-Based Heavy Metal Biosorbents: Enhanced Uptake of Cadmium and Mercury by Escherichia coli Expressing a Metal Binding Motif. Applied and Environmental Microbiology, October 1998, p. 4068-4072, Vol. 64, No. 10
This part codes for a peptide containing a metal binding motif. De novo design allows this peptide to have high affinity for metals. Affinity derives from Cys-Gly repeats. This peptide encodes two Cys-Gly-Cys-Cys-Gly repeats. Other metals besides arsenic, such as Cd and Hg, should be bound as well.
This peptide only contains two repeats of the metal binding motif due to size limitations when ordering oligos. Further repeats of the binding motif may enhance its function.
Copper Storage protein 1 (Csp1) is a tetrameric copper storage protein found in the periplasm of Methylosinus trichosporium OB3b. We investigated whether this part could act as a copper chelator when expressed in E. coli. We modified the protein by adding a TAT signal peptide from the E. coli enzyme CueO in place of the native TAT sequence and added a c terminal sfGFP fluorescence protein with a C terminal his tag in an attempt to direct the protein to the periplasm. The Csp1 and sfGFP are separated by a short hydrophilic, flexible linker.
Codon optimised for E. coli. linker added in order to allow the Csp1 and sfGFP to ideally fold separately. Hexahistidine purification tag on c-terminus to maintain TAT sequence.
CBDcex fused to phytochelatin (PC) EC20 with LacI promoter
BBa_J23105 was designed by John Anderson and BBa_B0034 was designed by Vinay S Mahajan, Voichita D. Marinescu, Brian Chow, Alexander D Wissner-Gross and Peter Carr IAP, 2003. This sequence was chemically synthesized.
This part codes costitutive promoter(BBa_J23105) and RBS(BBa_B0034). Most parts were expressioned by this part in our project.
Conversion of the iGEM parts registry to SBOL2.1