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)
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.
Ptac-NiCoTB, improves nickel and cobalt internalization
This part comprises a strong E. coli promoter with...
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.
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
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 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).
MerP, mercuric transport protein periplasmic component
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).
Source organism: Serratia marcescens. Plasmid pDU1358 kindly donated by Anne O. Summers.
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.
iGEM Calgary 2013 from a synthesized codon optimized construct
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).
MymT was cloned from pGB68 (kindly provided by Ben Gold, Cornell University), the gene's origin is the genome of ''M. tuberculosis''
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.
This parts encodes a protein capable of binding ferric ion. The protein includes a signal peptide targeting it to periplasmic space.
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.
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.
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.
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.
Source organism: Serratia marcescens. Amplified from plasmid pDU1358, kindly donated by Anne O. Summers.
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.
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.
RBS + GST (glutathione-S-transferase)-CRS5 (metallothionein) + Ter
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.
This part is BBa_K1460001 with the T7 promoter removed.
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.
Coding for the trans-acting regulator 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.
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.
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.
Mycobacterium tuberculosis. Ordered as codon optimised DNA.
pT7 + RBS + GST (glutathione-S-transferase)-CRS5 (metallothionein) + Ter
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
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.
pCopA MymT with divergent expressed CueR
pCopA MymT with plasmid constitutively expressed CueR
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.
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.
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.
CDS7 cadmium sulfate binding peptide coding sequence
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.
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.
Anderson Promoter + nixA + Ter + pT7 + GST-CRS5 + Ter
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
This part is a composite of BBa_K1460003 and BBa_K1460001.
BmtA (Lead/Zinc-binding metallothionein from Oscillatoria brevis, codon optimized for E. coli)
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.
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).
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.
The gene was cloned from pET29a-SmtA (kindly provided by Kevin Waldron, Newcastle University), it's origin is from the genome of ''Synechococcus PC''
We have to optimizes its codons in Arabidopsis Thaliana and removed the restriction sites for EcoR1, Xba1, Spe1 and Pst1.
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.
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.
pCopA MymT sfGFP with plasmid constitutively expressed CueR
Strong RBS + merA (mercuric ion reductase)+ terminator (BBa_ B0015)
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.
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.
CP47 His-tagged with Lac Promoter synthesized by IDT DNA Inc.
The MerB gene codes for the proetin which function is to cleave the organic part of the molecule of methyl-mercury
Inducible High Expression His-Tagged Photosystem II CP47 subunit
Anderson Promoter + CBP4 + pT7 + GST-CRS5 + Ter
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.
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).
This part is a composite of the parts BBa_K1460005 and BBa_K1460001.
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
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.
Gold binding peptides (GBP) is a kind of peptide which can bind on gold, usually used to immobilize protein on gold surface.
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.
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.
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.
Bacterioferritin from E. coli Nissle 1917 genomic DNA. Amplified by using PCR and cloned into pQE80L
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.
The OprF gene is from Pseudomonas aeruginosa.
merB, Organomercurial Lyase from Serratia marcescens
Amplified from pDU1358, kindly donated by Anne O. Summers.
RBS (B0034) + MerR (mercury-responsive transcription factor)
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.
The coding sequence of MerR comes from Tn, prefixed by an RBS part BBa_B0034 from Partsregistry
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.
Naoki Saito,Daiki Haraguchi,Yoshiharu Otaki,Ryuhei Minei
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).
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
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.
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.
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.
SmtA is a metallothionein that can bind to metal ions such as Cd(II).
This part is put together with Double terminator.
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.
This protein was selected from a pIII phage display library based on its ability to nucleate ZnS or CdS nanocrystals.
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 .
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.
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.
Arabidopsis thaliana (Columbia; leave) Pseudomonas Putida KT2240 (synthsized)
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.
Sequenced sourced from Professor David Wilson, of the Department of Molecular Biology and Genetics, Cornell University.
pCopA TAT Csp1 sfGFP with divergent CueR
pCopA TAT Csp1 sfGFP with plasmid constitutively expressed CueR
Anderson Promoter + merT + merP + Ter + pT7 + GST-CRS5 + Ter
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.
This part is a composite of parts BBa_K1460004 and BBa_K1460001
Regulation and transport of mercury ions
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!
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