PETase (PET-degrading enzyme, origin I. sakaiensis)
PETase is the PET-degrading enzyme first identified in Ideonella sakaiensis. This sequence is the E. coli K12 optimized DNA sequence for PETase.
This sequence was optimized for E. coli K12 using the Codon Optimization Tool from the IDT website.
This part comes from Ideonella sakaiensis, identified in "A bacterium that degrades and assimilates poly(ethylene terephthalate)" (Yoshida et al. 2016).
When used in conjunction with BisdA this enzyme can be used to degrade Bisphenol A. Activity has previously been recorded in E.coli by Miho Sasaki1 et al. 2005. Miho Sasaki1, Jun-ichi Maki, Ko-ichi Oshiman, Yoshinobu Matsumura1, and Tetsuaki Tsuchido.2005, Biodegradation of bisphenol A by cells and cell lysate from Sphingomonas sp. strain AO1. Biodegredation 16: 449-459
When used with BisdA the degredation of Bisphenol A sharply increases
Arabinose controlled protein degradation device
The purpose of this system is to control the expression of sspB, the degradation controller. The system is inducible by arabinose, therefore we can alter the arabinose levels to regulate the degradation of proteins with sspB degradation tag
sspB Biobrick is inserted after araR promoter+rbs system
Fusion protein of NADP+ Oxidoreductase and BisdA and BisdB, polycistronic
Fusion protein of NADP+ Oxidoreduktase and BisdA and BisdB, policistronic
We use this part to construct the degradation pathway of dioxin. This is a standard parts. Acoording to the standard, we add EcoR1,Not1 and Xba1 upstream; and spe1, Not1, Pst1 and stop condon downstream. Anyone of the five sites could be used . The vector is the standard vector,pSB1AC3, provided by biobrick08. We recommend double digestion of EcoRI+PstI.Digest in NEBuffer EcoRI + BSA at 37??C for about 3h.
an extradiol dioxygenase from the dibenzofuran- and dibenzo-p-dioxin-degrading bacterium Sphingomonas sp. strain RW1. Type: genomic DNA
Luna Barroco de Lacerda, Maria Clara Tavares Astolfi
The biorremediator device is composed by our ???essential biobrick??? (BBa_K1355001) + a MerA gene (BBa_K1355000). In the presence of mercury, The MerR protein will bind to it and MerT, MerP and MerA protein will be expressed and bioremediation will start! The MerP and MerT proteins are responsible for the transport of mercury from the periplasm to the cytoplasm, leading him to the mercury ion reductase (MerA)! The beautiful MerA be responsible for the reduction from Hg2+ in Hg0, which is volatile at room temperature and able to passively leave out the bacteria.
For this construct, we followed these steps: Extraction of plasmid DNA and DNA quantification of bacteria transformed with the Essential Biobrick (BBa_K1355001) and the bacteria transformed with the MerA Biobrick (BBa_E0840); Verifying the electrophoretic profile of the extracted plasmid DNA; Restriction enzyme digestion for Essential Biobrick with SpeI and PstI and for merA Biobrick with PstI and XbaI; Checking the electrophoretic profile of digested samples; Fragment of interest purification from the gel; Ligation the gel purification of merA gene and digested Essential Biobrick; Transformation of ligation in DH5-alpha; Plasmid DNA extraction from bacteria transformed; Check the electrophoretic profile to see results of samples linked (no fragments); Digestion of Essential Biobrick + merA Biobrick with EcoRI and PstI, aiming to analyze the fragment size to be isolated; Checking the electrophoretic profile of the digested sample to obtain results showing that the isolated fragment is the junction of Essential Biobrick +merA Biobrick ;
one part of the initial dioxygenase of the dioxin degradation pathway
The initial dioxygenase of the dioxin degradation pathway is a three-component enzyme with an atypical electron supply system.dxnA is one part of this muticomponent complex which acts as ring hydroxylase.dxnA harbors two subunits,dxnA1 and dxnA2. This is a standard parts. Acoording to the standard, we add EcoR1,Not1 and Xba1 upstream; and spe1, Not1, Pst1 and stop condon downstream. Anyone of the five sites could be used except pst1. The vector is the standard vector provided by biobrick08. We recommend double digestion of EcoRI+SpeI.Digest in NEBuffer EcoRI + BSA at 37??C for about 3h.
We use this part to construct the degradation pathway of dioxin.
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.
Promoter (luxR & HSL regulated -- lux pR)
Promoter activated by LuxR in concert with HSL The lux cassette of V. fischeri contains a left and a right promoter. The right promoter gives weak constitutive expression of downstream genes.This expression is up-regulated by the action of the LuxR activator protein complexed with the autoinducer, 3-oxo-hexanoyl-HSL. Two molecules of LuxR protein form a complex with two molecules of the signalling compound homoserine lactone (HSL). This complex binds to a palindromic site on the promoter, increasing the rate of transcription.
This promoter is based on the Vibrio fischeri quorum sensing gene promoters. Two genes LuxI and LuxR and transcribed in opposite directions as shown below. The original sequence from which the parts <bb_part>BBa_R0062</bb_part> and <bb_part>BBa_R0063</bb_part> were derived is shown in the picture below. <img src="<bb_file>Image1.gif</bb_file>" width="614" height="362">
Vinay S Mahajan, Voichita D. Marinescu, Brian Chow, Alexander D Wissner-Gross and Peter Carr
xccl laccase from Xanthomonas campestris with T7 promoter, RBS and His-tag
xccl laccase from Xanthomonas campestris with pT7 and HIS tag
Xanthomonas campestris pv. campestris B100
This is the DNA sequence of a Y-type hammerhead ribozyme that targets and cleaves the PenI repressor (Part BBa_C0074). This ribozyme has target-binding flanking arms adjacent to the Y-type hammerhead catalytic core and synthetic extended arms located towards the 5' and 3' ends. The catalytic core allows for the sequence-specific cleavage of sites within the coding sequence of PenI. Hammerhead ribozymes are able to cleave at any NUH within an RNA sequence, where H represent any nucleotide except guanine (Mir 2001), although with GUC as the hammerhead consensus cleavage site (Lieber, 1995). The binding of the flanking arms adjacent to the hammerhead catalytic core favours the recognition and cleavage of only one target site. Additional secondary structure within the flanking arms was detected to a small degree by using IDT OligoAnalyzer's Hairpin tool (Owczarzy 2008) as is depicted below. [image] The synthetic extended arms are designed to bind to the complementary extended arms of another similar PenI-cleaving ribozyme (Part _____), but do not contribute to any additional secondary structure as confirmed via the Hairpin tool in IDT's OligoAnalyzer (Owczarzy 2008), as both arms of a particular ribozyme only contain a purine and pyrimidine pair that do not bind to each other (in this case, A and G). The two-nucleotide extended arms are also staggered in number to avoid slippage in binding and bind to its complement pair at significantly higher melting temperatures than the adjacent flanking arms. This feature allows for the sequestering of the two ribozymes when both are present and significantly decreasing any ribozyme activity. Regions of complementarity between the two target-binding regions of both ribozymes are also present to ensure that the functional catalytic cores are not able to cleave their mRNA targets, even when bound. The characteristics of this expressed pair of ribozymes were considered to create a functional RNA-based XOR logic gate. References Mir, A. A., Lockett, T. J. & Hendry, P. Identifying ribozyme-accessible?? sites using NUH triplet-targeting gapmers. Nucleic Acids Res 29, 1906???1914 (2001). Lieber, A. & Strauss, M. Selection of efficient cleavage sites in target RNAs by using a ribozyme expression library. Mol. Cell. Biol. 15, 540???551 (1995). Owczarzy, R. et al. IDT SciTools: a suite for analysis and design of nucleic acid oligomers. Nucleic Acids Res 36, W163???W169 (2008).
Although this particular part is likely to only cleave the PenI repressor mRNA due to its sequence specificity, the simple in silico design of this ribozyme allows for the cleavage of virtually any RNA sequence containing a cleave site (NUH). This particular sequence was obtained by first scanning our target PenI mRNA for the optimal cut site GUC. The 10 nucleotides surrounding this cut site were obtained and the complement of this area was generated for the ribozyme flanking arms. The length of this region was decided by ideal melting temperature (51.4 degrees Celsius). Additional synthetic extended arms were subsequently added to the 5' and 3' ends of the sequence for binding to the complement ribozyme in an XOR gate. This region of non-pairing purines and pyrimidines was staggered in base repeats to avoid slippage of binding and was defined in length by significantly higher melting temperatures (approximately 68 degrees Celsius). Finally, the catalytic core of the Y-type hammerhead ribozyme was introduced at the appropriate region. Throughout this entire process, secondary was continually checked and minimized using IDT OligoAnalyzer's Hairpin tool (Owczarzy 2008). The complete ribozyme sequences was also BLASTed again the E.coli genome, which was our chassis and yielded insignificant results. Other factors such as differential length or target binding regions due to varying melting temperature and GC content were also considered during the in silico design of this ribozyme and its pair in the XOR logic gate. References Owczarzy, R. et al. IDT SciTools: a suite for analysis and design of nucleic acid oligomers. Nucleic Acids Res 36, W163???W169 (2008).
The target-binding flanking arms of the ribozyme were obtained from the complement of the PenI Repressor sequence.
Site directed mutagenesis to remove internal EcoRI site
katE (catalase) from E. coli. Site directed mutagensis to remove an EcoRI site, and an added C-term ssrA (LAA) tag for fast cell clearance.
Removal of an EcoRI site by site directed mutagenesis and addition of an ssrA tag with PCR primers
E. coli genomic PCR and addition of ssrA tag with primers during PCR
Xenobiotic Reductase B, created for use as a Trinitrotoluene and Nitroglycerine degrading protein. A monomeric flavin that can reduce certain nitro- groups to nitrate-. Increases the resistance of organisms to the toxic effects of nitrocompounds. This sequence encodes for a protein (with an attached His (x6) Tag to allow for purification) and nothing else. It therefore requires regulatory sequences (promoter/RBS/terminator) to be added for expression.
The original sequence of this part was found using the NCBI database and taken from Pseudomonas putida BIRD-1. The sequence was then codon-optimised for increased expression in E. coli. This has resulted in a novel synthetic sequence.
Source of original sequence: Pseudomonas putida BIRD-1.
Exo- and endo- glucanases from the cellulolytic bacterium Cellulomonas fimi under the E. coli lac promoter. Together with betaglucosidases, the bacterium degrades cellulose into D-glucose.
Cex is a GC rich part and may require extended denaturation in amplifying the gene.
Kwabena O. Duedu, Chris French, Sahreena Lakhundi
Exo- and endo- glucanases from the cellulolytic bacterium Cellulomonas fimi under the E. coli spac promoter (BBa_J15506). Together with betaglucosidases, the bacterium degrades cellulose into D-glucose.
Cex is a GC rich part and requires extended denaturation with glycerol or commercial GC enhancers
Fusion protein of BisdA and BisdB behind constitutive promoter
Transcriptional terminator consisting of a 64 bp stem-loop.
This part has been designed to allow the presence/absence of TetR to control the production of BisdA
This part has been designed to allow the presence/absence of TetR to control the production of BisdB
2，2???，3-Trihydroxybiphenyl 1,2-dioxygenase. The second step enzyme in dixon degradati
2，2???，3-Trihydroxybiphenyl 1,2-dioxygenase.The second step meta cleavage enzyme in dioxin degradation pathway. According to the standard, we add EcoR1,Not1 and Xba1 upstream; and spe1, Not1, Pst1 and stop condon downstream. Anyone of the five sites could be used except Not1. The vector is the standard vector provided by biobrick08. We recommend double digestion of EcoRI+SpeI.Digest in NEBuffer EcoRI + BSA at 37??C for about 3h.
We use it to construct the degradation pathway of dioxin. According to the standard, we add EcoR1,Not1 and Xba1 upstream; and spe1, Not1, Pst1 and stop condon downstream. Anyone of the five sites could be used except Not1. The vector is the standard vector provided by biobrick08. We recommend double digestion of EcoRI+SpeI.Digest in NEBuffer EcoRI + BSA at 37??C for about 3h.
from the dibenzofuran- and dibenzo-p-dioxin-degrading bacterium Sphingomonas sp. strain RW1. Type: genomic DNA
tthl laccase from Thermus thermophilus with T7 promoter, RBS and His-tag
tthl (Laccase from Thermus thermophilus) with T7, RBS and HIS tag
Endoglucanase A (CenA) from the cellulolytic bacterium Cellulomonas fimi (accession M15823) used in the degradation of cellulose into cellobiose, before using beta-glucosidase to catalyse the conversion of cellobiose to D-glucose.
YebF + PETase (PET-degrading enzyme, origin I. sakaiensis)
This GGS linker is a linker sequence that neutrally connects two protein domains together. The linker is composed of repetitions of glycine-glycine-serine, which are known to be neutral and not interfere with the rest of the protein. This is an alternate version of BBa_K2010002, but serves the same function.
The DNA sequence has been optimized for E. coli K12 and modified to facilitate gBlock synthesis, by avoiding repeated sequences.
This sequence is publicly known and was privately modified.
Pspac hybrid promoter for Bacillus subtilis
Amplified from plasmid pVK168. The original reference is: Yansura DG and Henner DJ, 1984, Use of the Escherichia coli lac repressor and operator to control gene expression in Bacillus subtilis, PNAS USA 81, 439-443.
This is the Pspac promoter from pVK168. It is a hybrid promoter based on a B.subtilis phage promoter with the control elements from the E. coli lac promoter. It is controllable in B. subtilis provided lacI is also expressed. Apparently it is also functional in E. coli.
The Estrogen Responsive Element was taken from the Human Estrogen Responsive Element
This composite part contains LacIQ (BBa_I14032) and Estrogen Receptor Element (ERE) and TetR (BBa_C0040). The Estrogen Responsive element is to be used in conjunction with ER (Estrogen Receptor). This design involves ER binding to the ERE and disrupting the constituative promoter LacIQ from producing TetR via physically getting in the way of the polymerase.
There is some data that suggests the ER is toxic to the cell. However in our experiments while we did see a decrease in growth rate we did not find ER to be fatal to the cell.
pelB + PETase (PET-degrading enzyme, origin I. sakaiensis)
This GGS linker is a linker sequence that neutrally connects two protein domains together. The linker is composed of repetitions of glycine-glycine-serine, which are known to be neutral and not interfere with the rest of the protein.
Long-Chain Alkane Monooxygenase of Geobacillus thermodinitrificans.
A construction intermediate used to produce BBa_K338003 . Contains the first of three genes required to synthesize polyhydroxybutyrate (PHB).
Designed to be ligated upstream of BBa_K338023 and BBa_K338024 .
The Registry of Standard Biological Parts
pNB-Est 13: Esterase PET cleaving enzyme
Marie Burghard, Henrik Cordes, Jascha Diemer, Adrian Eilingsfeld, Sven Jager, Philipp Rottmann, Rene Sahm, Arne Wehling
The pNB-ESterase 13 is a mutant derived from pNB-Esterase from Bacillus licheniformis. It showed the highest affinity and activity towards PET.
Our team obtained it from Henkel GmbH on an pET26b(+) vector: Fakult, M., & Enzymtechnologie, M. (2006). Isolierung, Identifizierung und biochemische Charakterisierung Dialkylphthalat spaltender Esterasen.
cex coding sequence encoding Cellulomonas fimi exoglucanase
Purified ''Cellulomonas fimi'' genomic DNA.
The fungus ''Cellulomonas fimi'' uses an exoglucanase (from ''cex'') along with 3 endoglucanases in the degradation of cellulose into cellobiose, before use B-glucosidase to catalyse the conversion of cellobiose to D-glucose.
The original sequence had a PstI site for 781-786. We added the silent mutation CAG->CAA (Gln) by MABEL to remove this site.
A construct created to degraded TNT and nitroglycerine. The construct contains the coding sequence for XenB (BBa_K1398000), an enzyme with the capability to degrade nitro- groups in chemicals. The construct also contains an inducible promoter (BBa_R0010), a strong RBS (BBa_B0034) and a double terminator (BBa_B0010). The protein has been codon-optimised for expression in E. coli.
This construct was created to allow for the over-expression of XenB, so it contains high expression regulatory sequences. A lactose inducible promoter has been used to give control over expression.
The original source of XenB was Pseudomonas putida. The regulatory sequences were acquired from the iGEM repository.
Marie Burghard, Henrik Cordes, Jascha Diemer, Sven Jager, Philipp Rottmann, Rene Sahm, Arne Wehling
RBS-PhoA-His6tag-pNBEst13-Myctag-EstA is a long name for a long protein construct. It is a fusion protein which is secreted into the peri plasma in order to be integrated into the outer membran. When the phoA signal sequence is cut off, the N-terminus is formed by our pNB-Est13. This esterase shows actitivty towards PET and is anchored onto the cell by its inactive EstA membrane anchor.
We got it synthesized by GENEART but added the RBS via PCR.
This is no natural or even native construct. We got it synthesized by GENEART. but added the RBS via PCR.
Tetracycline resistance protein from Bacteroides fragilis
Tet X monooxygenase: Characterization: In vivo qualitative experiements：
protein sequence--gene sequence(coodon preferance) 3 synonynous mutaions
Tetracycline resistance protein from transposon Tn4351/Tn4400 found in Bacteroides fragilis
bhal laccase from Bacillus halodurans with T7 promoter, RBS and His-tag
bhal laccase from Bacillus halodurans with T7 and HIS
tphB: reaction from DCD to protocatechuate
We used a linker attached to a protein membrane "estA"
ecol laccase from E. coli with T7 promoter, RBS and His-tag
E.coli laccase ORF with T7, RBS and HIS tag
The DNA was amplified from E. coli BL21 genomic DNA using primers based on published sequence (Genbank accession J01636, gi:146575). The annotation shown here is based on that associated with this Genbank entry. The sequence shown here is derived by sequencing the construct.
This part (submitted in pSB1A2) consists of the lac promoter and lacZ' gene, encoding the N-terminal 76 amino acid residues of LacZ, sufficient to complement the lacZ-delta-M15 mutation for blue-white selection on Xgal plates. A SacI site has been introduced at the 3' end, overlapping the XbaI site of the Biobrick prefix. This is designed to be used as a cloning vector for making new biobricks. PCR primers can be designed with a SacI site in one primer and an SpeI site in the other. This removes the necessity for an excessively long non-complementary tail on one primer bearing either the full biobrick prefix or suffix. The PCR product can then be digested with SacI and SpeI for insertion into this plasmid, replacing the Plac-lacZ' cassette. Recombinant plasmids will then be white on IPTG/Xgal plates, whereas any that still contain the original insert will be blue. We have used this strategy to prepare several biobricks, including BBa_J33204, which contains the xylE gene encoding catechol-2,3-dioxygenase. (For making biobricks that contain lacZ', BBa_J33204 can be used in the same way; in this case, clones with plasmids that still contain xylE will turn yellow on addition of a drop of 10 mM catechol.)
Note that the SacI site overlaps the SpeI site. The Biobrick prefix ends ...TCTAGAG. When this is added to the CTC at the start of the sequence shown here, the SacI site, GAGCTC, is generated.
A device that expresses the XylE- catechol 2,3-dioxygenase to degrade the catechol .
Before the XylE - catechol 2,3-dioxygenase, the promoter J23100 and the RBS B0034 are connected. The device can be used to degrade the catechol in some degradation system to decrease the content of the catechol for the catechol can restrain the growth of the bacteria, also, the catechol is a kind of common degradation intermediate product.
We linked the K316003 with the promoter J23100 and the RBS B0034 ahead.
We use the parts in kit2016 to complete the device.
bphD codes 2-hydroxy-6-oxo-6-phenylhexa-2,4- dienoate hydrolase
bphD codes 2-hydroxy-6-oxo-6-phenylhexa-2,4- dienoate hydrolase, which is the forth step enzyme of PCBs degradation pathway. This is a standard parts. Acoording to the standard, we add EcoR1,Not1 and Xba1 upstream; and spe1, Not1, Pst1 and stop condon downstream. Anyone of the five sites could be used except EcoR1. The vector is the standard vector, pSB1AC3, provided by biobrick08. We recommend double digestion of xbaI+speI.Digest in NEB Buffer 2 + BSA at 37??C for about 3h.
We use this part to construct the degradation pathway of PCBs This is a standard parts. Acoording to the standard, we add EcoR1,Not1 and Xba1 upstream; and spe1, Not1, Pst1 and stop condon downstream. Anyone of the five sites could be used except EcoR1. The vector is the standard vector, pSB1AC3, provided by biobrick08. We recommend double digestion of xbaI+speI.Digest in NEB Buffer 2 + BSA at 37??C for about 3h.
From the PCBs-degrading bacterium Burkholderia xenovorans LB400 chromosome 3. Type: genomic DNA
tthl laccase from T. thermophilus with constitutive promoter J23100, RBS and His-tag
tthl laccase (T. thermophilus) with constitutive promoter and HIS tag
This part codes for the enzyme Lignin Peroxidase which initiates the breakdown of lignin, a complex biopolymer in biomass. The part is currently in testing, but should produce the active enzyme. It can be used in bioenergy projects to begin the process of making biofuels. Also, there has been some discussion about the possibility of using peroxidases to break down synthetic polymers.
This part is one of a family of highly homologous genes in P.C. It was nearly impossible to isolate the gene using primers specific for the coding sequence. It was necessary to use a sequential set of PCR primers specific for the upstream and downstream regions and then the specific coding sequence, respectively.
cDNA of Phanerochaete Chrysosporium grown on Aspen Wood Culture at University of Wisconsin/USDA Forest Products Lab.
Fusion protein of S-Layer SgsE with T7/lac and BisdA and BisdB
ompT + PETase (PET-degrading enzyme, origin I. sakaiensis)
MHETase is the MHET(Mono(2-hydroxyethyl) terephthalate)-degrading enzyme first identified in Ideonella sakaiensis. This sequence is the E. coli K12 optimized DNA sequence for MHETase.
AraC+Pbad promoter+RBS+signal peptide phoA+Cellulase+Linker+estA membrane protein
The composite part includes a functional expression vector for autotransporter membrane protein estA, with a linker united to a cellulase, attached to a membrane signaling peptide. This was our final construct missing a terminator
When used in conjunction with BisdB this enzyme can be used to degrade Bisphenol A. Activity has previously been recorded in E.coli by Miho Sasaki1 et al. 2005. Miho Sasaki1, Jun-ichi Maki, Ko-ichi Oshiman, Yoshinobu Matsumura1, and Tetsuaki Tsuchido.2005, Biodegradation of bisphenol A by cells and cell lysate from Sphingomonas sp. strain AO1. Biodegredation 16: 449-459
These parts where designed to include a his tag to make it easier to view enzyme presence
Fusion protein of SbpA, NADP+ Oxidoreductase, BisdA and BisdB with PT7/lac
PT7 + RBS + SbpA || Red || BisdA || BisdB
Double terminator consisting of BBa_B0010 and BBa_B0012
pelB + sfGFP + PETase (PET-degrading enzyme, origin I. sakaiensis)
This part when used will allow the production of TetR to increase the production of BisdA and BisdB. When produced BisdA and BisdB will cause the degredation of Bisphenol A.
NADP+ Oxidoreductase and polycistronic BisdA and BisdB
NADP+ Oxidoreduktase and polycistronic BisdA and BisdB
Aldehyde dehydrogenase from Geobacillus thermoleovorans B23.
Nucleotide sequence optimized for expression in E.coli.
Aiia is a protein that degrades autoinducer(acyl HSL).In this part production of Aiia can be regulated by varying aTc concentration in the medium.
ompT + sfGFP + PETase (PET-degrading enzyme, origin I. sakaiensis)
This His-tag is a sequence that allows a conjunct protein to be purified. The tag is composed of repetitions of histidines. This is an alternate version of BBa_K2010004, but serves the same function.
autoinducer inactivation enzyme, AaiiA (+RBS)
PC and AraC from the AraBAD operator. PC and AraC are on the complementary strand, reading right to left.
Cellulase with a mutation, giving it a smalle km
It also has a SacI site and a NheI site after prefix, and before suffix EcorI XbaI SacI Cellulase NheI SpeI PstI
autoinducer inactivation enzyme from Bacillus; hydrolyzes acetyl homoserine lactone
AF196486 from Bacillus sp. 240B1 putative metallohydrolase (aiiA) gene. Dong,Y.H., Xu,J.L., Li,X.Z. and Zhang,L.H.: AiiA, an enzyme that inactivates the acylhomoserine lactone quorum-sensing signal and attenuates the virulence of Erwinia carotovora, Proc. Natl. Acad. Sci. U.S.A. 97 (7), 3526-3531 (2000).
Coding region for the autoinducer inactivation enzyme A (aiiA) LVA tagged. The gene was originally isolated from Bacillus sp. 240B1 and it encodes an enzyme that catalyzes the degradation of N-acyl homoserine lactones (AHLs)--quorum sensing autoinducers.
References (unparsed) here: Dong,Y.H., Xu,J.L., Li,X.Z. and Zhang,L.H.: AiiA, an enzyme that inactivates the acylhomoserine lactone quorum-sensing signal and attenuates the virulence of Erwinia carotovora, Proc. Natl. Acad. Sci. U.S.A. 97 (7), 3526-3531 (2000). http://www.pnas.org/cgi/content/full/97/7/3526 Lee SJ, Park SY, Lee JJ, Yum DY, Koo BT, Lee JK.: Genes encoding the N-acyl homoserine lactone-degrading enzyme are widespread in many subspecies of Bacillus thuringiensis, Appl Environ Microbiol 2002 Aug;68(8):3919-24. http://aem.asm.org/cgi/content/full/68/8/3919?view=full&pmid=12147491 References (unparsed) here: Dong,Y.H., Xu,J.L., Li,X.Z. and Zhang,L.H.: AiiA, an enzyme that inactivates the acylhomoserine lactone quorum-sensing signal and attenuates the virulence of Erwinia carotovora, Proc. Natl. Acad. Sci. U.S.A. 97 (7), 3526-3531 (2000). http://www.pnas.org/cgi/content/full/97/7/3526 Lee SJ, Park SY, Lee JJ, Yum DY, Koo BT, Lee JK.: Genes encoding the N-acyl homoserine lactone-degrading enzyme are widespread in many subspecies of Bacillus thuringiensis, Appl Environ Microbiol 2002 Aug;68(8):3919-24. http://aem.asm.org/cgi/content/full/68/8/3919?view=full&pmid=12147491 BBa_C0060 insert contains open reading frame (nucleotides 49-801) of the GeneBank sequence AF196486 followed by the LVA tag and two double stop codons inserted in the BioBrick prefix and suffix flanking regions. The original stop codon was TAG and in the present sequence it was substituted by TAATAA.
Vinay S Mahajan, Brian Chow, Peter Carr, Voichita D. Marinescu and Alexander D. Wissner-Gross
Alcohol dehydrogenase from Bacillus thermoleovorans B23.
autoinducer inactivation enzyme aiiA (no LVA)
Fusion protein of NADP+ Oxidoreductase and BisdA and BisdB
Fusion protein of NADP+ Oxidoreduktase and BisdA and BisdB; RFC 25
The sequence was obtained by applying all mutations that upregulated AraC binding and subsequent promoter activity listed in reference [1].
pBAD is an E.coli promoter that is induced by L-arabinose. K206000 is a mutagenized pBAD promoter that is responsive to lower concentrations of arabinose than wild type ( I13453 ) and, additionally, exhibits a higher maximum of protein expression as measured by coupling to a fluorescent reporter.
There were no special design considerations.
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.
2,3-dihydroxy-2,3-dihydrophenylpropionate dehydrogenase, the second step enzyme in PCBs degradation
bphB codes 2,3-dihydroxy-2,3-dihydrophenylpropionate dehydrogenase, the second step enzyme in PCBs degradation pathway. Acoording to the standard, we add EcoR1,Not1 and Xba1 upstream; and spe1 Not1, Pst1 and stop condon downstream. The initial dioxygenase of the dioxin degradation pathway is a three-component enzyme with an atypical electron supply system.dxnA is one part of this muticomponent complex which acts as ring hydroxylase.dxnA harbors two subunits,dxnA1 and dxnA2. This is a standard parts. Acoording to the standard, we add EcoR1,Not1 and Xba1 upstream; and spe1, Not1, Pst1 and stop condon downstream. Anyone of the five sites could be used. The vector is the standard vector pSB1AC3 provided by biobrick08. We recommend double digestion of EcoRI+PstI.Digest in NEBuffer EcoRI + BSA at 37??C for about 3h.
We use it to construct the degradation pathway of PCBs. This is a standard parts. Acoording to the standard, we add EcoR1,Not1 and Xba1 upstream; and spe1, Not1, Pst1 and stop condon downstream. Anyone of the five sites could be used. The vector is the standard vector pSB1AC3 provided by biobrick08. We recommend double digestion of EcoRI+PstI.Digest in NEBuffer EcoRI + BSA at 37??C for about 3h.
From the plasmids pAIA73 provided by Professor Bernd Holfer from Department of Microbiology, Gesellschaftfiir Biotechnologische Forschung, D-381 24 Braunschweig, Germany. Type: genomic DNA
Coding sequence for Nuclease regulated by T7-promoter
Usage: Nuclease is an enzyme with the capability of cleaving phosphodiester bonds between nucleotides in DNA. This enzyme can be used in applications concerning degradation of DNA. In our project the aim was to evaluate and use Nuclease degrading abilities to inhibit the biofilm formation produced by Staphyloccus Auerus. The encoding part of the BioBrick is derived from BBa_K729004, made by iGEM London 2012. The T7 promoter (BBa_k525998) has been inserted to control protein expession under IPTG induction. To regulate the protein expression under T7 promoter control, E.coli containing a T7 polymerase must be used, for instance BL21(DE3). Biology: Nuclease is an extracellular enzyme found in Staphyloccus Aureus. [3] S.Aureus encodes for two Nucleases, Nuc1 and Nuc2, [3] whereof the last-mentioned is a part of this BioBrick. Nuc2 is connected to the membrane with a N-terminal anchor. [3] There are several bacterial pathogens using Nuclease to combat the immune response in the host by for instance avoiding neutrophil extracellular traps. (NETs). [1] Furthermore, since eDNA is an important component of biofilm [2] Nuc is able to work as a regulator of biofilm formation. [2] Studies have suggested that the expression of Nuc contributes to the inability to produce biofilm [2] and during biofilm formation-conditions the expression of Nuc is repressed. [3]
The subparts was aligned by 3A Assembly.
We used Rybosome Binding Site BBa_B0034 designed by by Vinay S Mahajan, Voichita D. Marinescu, Brian Chow, Alexander D Wissner-Gross and Peter Carr, evaluated by Warsaw's 2010 team. we added membrane protein signal peptide phoA and cellulase immediately next to it
Since this part is supposed to be attached to part BBa_K633001 in order to geerate part BBa_K633014, we had to begin with both the signal peptide and then the extracelular domain, which is located on the N terminal side of the polypeptide chain.
Lutz, R., Bujard, H., Nucleic Acids Research (1997) 25, 1203-1210.
Sequence for pTet inverting regulator driven by the TetR protein.
BBa_R0040 TetR-Regulated Promoter is based on a cI promoter. It has been modified to include two TetR binding sites and the BioBrick standard assembly head and tail restriction sites.
June Rhee, Connie Tao, Ty Thomson, Louis Waldman
bpul (laccase from Bacillus pumilus) with T7 promoter, RBS and HIS tag
bpul (Laccase from Bacillus pumilus) with T7, RBS and HIS tag
This Construct will allow for the detection and degredation of BPA in cells already containing the ER (K123003) biobrick. It works in two systems first. Together K123003 and K123009 produce a NOT gate where when Bisphenol A (BPA), Estrogen or any other estrogen mimic is in the system the product of K123003 dimerizes and attaches to the ERE site of K123009. This stops the production of TetR (R0040) by phsically stoping the Polymerase from transcribing it. This allows the pTet(C0040)promoter to turn on the production of RFP (E1010) for detection as well as BisdA(K123000) and BisdB(K123001)which allow for the degredation of BPA.
While BisdA and BisdB have only been shown to degrade BPA we have put in RFP to allow for the detection of other estrogen mimics as well.
Autoinducer inactivation enzyme regulated by LuxR/HSL promoter
We made this construct with LuxR/HSL promoter R0062, B0034 RBS, and the C0060 aiiA gene in order to give us more control with our repressilator system. The aiiA gene will be turned on by the LuxR/HSL promoter in the presence of the LuxR activator protein complexed with the autoinducer 3-oxo-hexanoyl-HSL.
cenA coding sequence encoding Cellulomonas fimi endoglucanase A
The cellulolytic bacterium ''Cellulomonas fimi'' uses 3 endoglucanase (including CenA) and an exoglucanase in the degradation of cellulose into cellobiose, before use B-glucosidase to catalyse the conversion of cellobiose to D-glucose.
This part codes for an enzyme that catalyzes the synthesis of acetoacetyl-coenzyme A from two molecules of acetyl-coenzyme A. The enzyme can also act as a thiolase, catalyzing the reverse reaction and generating two-carbon units from the four-carbon product of fatty acid oxidation.
This part was codon-optimized for expression in E. coli. The following restriction enzyme sites were also removed: EcoRI, NotI, XbaI, SpeI, PstI, NheI, AvrII, ApoI, MfeI, NsiI, SbfI.
This coding region is native to ''Ralstonia eutropha'' H16. The original sequence was obtained from GenBank (VERSION NC_008313.1, GI:113866031).
Fusion protein of S-Layer SbpA with T7/lac and BisdA and BisdB
RBS.3 (medium) -- derivative of BBa_0030
Weak1 RBS based on Ron Weiss thesis. Strength is considered relative to <bb_part>BBa_B0030</bb_part>, <bb_part>BBa_B0031</bb_part>, <bb_part>BBa_B0033</bb_part>.
Varies from -6 to +1 region from original sequence to accomodate BioBricks suffix ("RBS-2" in figure 4-14 of thesis). Contact info for this part: Brian Chow
Cellulomonas fimi exoglucanase cex (accession M15824) with RBS
RBS + Exoglucanase (cex, BBa_K118022) from the cellulolytic bacterium Cellulomonas fimi (accession M15824).
GC rich part. Extended denaturation stage in PCR with glycerol or commercial GC rich enhancers may be required
bphC codes 2,3-Dihydroxybiphenyl-1,2-dioxygenase
bphC codes 2,3-Dihydroxybiphenyl-1,2-dioxygenase that is the third step enzyme in PCBs degradation pathway. This is a standard parts. Acoording to the standard, we add EcoR1,Not1 and Xba1 upstream; and spe1, Not1, Pst1 and stop condon downstream. Anyone of the five sites could be used. The vector is the standard vector, pSB1AC3, provided by biobrick08. We recommend double digestion of EcoRI+pstI.Digest in NEBBuffer EcoRI + BSA at 37??C for about 3h.
We use this part to construct the degradation pathway of PCBs.
strong synthetic E. coli ribosome binding site with SacI site.
This is a strong synthetic E. coli ribosome binding site. It is synthesised as two complementary oligonucleotides rather than being incorporated into a biobrick plasmid. It incorporates a SacI site overlapping the XbaI site, which is preserved when it is added to any other biobrick. This allows easy detection of the RBS after it has been added upstream of a biobrick coding sequence in a plasmid vector.
Note the presence of a SacI site overlapping the XbaI site, which is preserved when this biobrick is added to any other biobrick. At the time of writing, this biobrick is added as a short piece of DNA composed of two complementary oligonucleotides rather than being incorporated into a biobrick cloning vector by itself. It can be added upstream of a biobrick coding sequence, and its presence can easily be detected in miniprep DNA on a gel by using a SacI-SpeI or similar digest.
beta-glucosidase gene bglX (chu_2268) from Cytophaga hutchinsonii
Genome sequence of the cellulose-degrading Gram negative bacterium Cytophaga hutchinsonii.
This is a beta-glucosidase gene (bglX, locus chu_2268) from the genome sequence of the cellulose-degrading Gram negative bacterium Cytophaga hutchinsonii.
Polycistronic expression of BisdA and BisdB
This is one of the two catalases from E. coli.
Site directed mutagensis to remove an EcoRI site.
Marie Burghard, Henrik Cordes, Jascha Diemer, Adrian Eilingsfeld, Sven Jager, Rene Sahm, Arne Wehling
A cutinase is a cuticula degrading hydrolase from the fungus Fusarium solani pisi. It shows activity towards PET.
Surface Expression is very hard to do. FsC shows lipolytic activity and attacks the outer membrane of gramm negativ bacteria. This activity leads to a certain toxicity for expression hosts. In addition FsC needs to be expressed in the periplasm due to folding issues.
Cloned from vector pEST100 from Becker et all 2005: (Becker, S., Theile, S., Heppeler, N., Michalczyk, A., Wentzel, A., Wilhelm, S., Jaeger, K.-E., et al. (2005). A generic system for the Escherichia coli cell-surface display of lipolytic enzymes. FEBS letters, 579(5), 1177???82. doi:10.1016/j.febslet.2004.12.087)
One part of the initial dioxygenase in dioxin degradation pathway
The initial dioxygenase of the dioxin degradation pathway is a three-component enzyme with an atypical electron supply system. RedA2 is one part of this muticomponent complex which acts as reductase. This is a standard parts. Acoording to the standard, we add EcoR1,Not1 and Xba1 upstream; and spe1, Not1, Pst1 and stop condon downstream. Anyone of the five sites could be used except pst1. The vector is the standard vector provided by biobrick08. We recommend double digestion of EcoRI+SpeI.Digest in NEBuffer EcoRI + BSA at 37??C for about 3h.
We use this part to construct the degradation pathway of dioxin. The initial dioxygenase of the dioxin degradation pathway is a three-component enzyme with an atypical electron supply system. RedA2 is one part of this muticomponent complex which acts as reductase. This is a standard parts. Acoording to the standard, we add EcoR1,Not1 and Xba1 upstream; and spe1, Not1, Pst1 and stop condon downstream. Anyone of the five sites could be used except pst1. The vector is the standard vector provided by biobrick08. We recommend double digestion of EcoRI+SpeI.Digest in NEBuffer EcoRI + BSA at 37??C for about 3h.
RedA2 is a reductase from the dibenzofuran- and dibenzo-p-dioxin-degrading bacterium Sphingomonas sp. strain RW1. Type: genomic DNA
A basic part required for the chaperone/toxicity device
{{BerkiGEM2007-Biobrick2.0Intro}} Construction of a Biobrick 2.0 basic part in plasmid pBca9145-Bca1144 (Clone # 5) for the HPI, katG open reading frame from Salmonella typhimurium LT2 PCR VU03F.katG /VU06R.katG on HPI, katG (1316 bp, gp = A) PCR VU05F.katG/VU04R.katG on HPI, katG (924 bp, gp = B) --------------------------------------------------- PCR VU03F/VU04R on A+B (2212 bp, BglII/XhoI) Digest pBca9145-Bca1144 (Clone # 5) (BglII/XhoI, 2063+910, L) Product is pBca9145-VU2 --------------------------------------------------- VU03F.katG (External oligo) ccaaaAGATCTatgagcacgaccgacgataccc VU04R.katG (External oligo) gctagCTCGAGttaGGATCCttattgcagatcgaaacggtcc VU05F.katG (Internal oligo) gccgaaagaagaCctgatctggcag VU06R.katG (Internal oligo) ctgccagatcagGtcttctttcggc
This is half of a planned part which would contain all three PHA synthase genes required to produce [http://en.wikipedia.org/wiki/Polyhydroxybutyrate polyhydroxybutyrate] (PHB) in cells: ''phaA'', ''phaB1'', ''phaC1''. This half contains only ''phaA'': BBa_K156012 . It was designed to be ligated upstream of part BBa_K338004.
When ligated upstream of BBa_K338004, the completed construct was designed to express all three PHA synthase genes required to make PHB oligomers from soybean oil. The three genes would be transcribed polycistronically on a single mRNA transcript under the IPTG-inducible control of the BBa_K215000 promoter. Naturally, each gene is preceded by a standard RBS ( B0034 ) and the transcript finishes with a strong terminator ( B0015 ), for a total size of about 3500bp. Note that these three genes should only cause the production of PHB ''oligomers'' in cells, not hardened plastic. A crosslinking agent is required to link the oligomers and form the final plastic product. Over-expression of the ''phaC1'' gene could cause some crosslinking, but this has not been experimentally verified.
**highly** engineered mutant of red fluorescent protein from Discosoma striata (coral)
Campbell et al., PNAS v99 p7877 URL
monomeric RFP: Red Fluorescent Protein. Excitation peak: 584 nm Emission peak: 607 nm
TAATAA double stop codon added (DE). Four silent mutations made to remove three EcoRI sites and one PstI site: A28G, A76G, A349G, G337A.
SspB protein targets proteins tagged with modified degradation tag, ssrA, and deliver them to ClpXP for proteolysis. Modified ssrA tags are fused onto the 3??? end of a gene.
Forward primer used: GATCTG-GAATTCGCGGCCGCTTCTAG-ATGGATTTGTCACAGCTAAC (Clamp sequence - Standard Biobrick prefix - first 20 base from the Biobrick) Reverse primer used: TGTGAC-ACTAGTA-TTACTTCACAACGCGTAATGC (Clamp sequence - SpeI site - last 21 base from the Biobrick)
498 bp long sspB CDS is amplified by PCR with dangling end primers with EcoRI-NotI-XbaI restriction site at 5??? and SpeI at 3??? and inserted into a Biobrick compatible vector. The sequence is taken from E. coli strain DH5 alpha. Genbank accession number for E. coli MG1655 strain is NC_000913.2
Fusion protein of SgsE, NADP+ Oxidoreductase, BisdA and BisdB with PT7/lac
PT7 + RBS + SgsE || Red || BisdA || BisdB ; RCU 25
Fusion protein of NADP+ Oxidoreductase and BisdA and BisdB with middle strong promoter,RBS
Fusion protein of ferredoxin-NADP+ oxidoreductase, BisdA and BisdB; RFC 25 (Freiburg BioBrick assembly standard) for characterization of intra- and extracellular BPA degradation.
tthl (Laccase from Thermus thermophilus) coding sequence
converts aliphatic medium-chain-length alcohols into aldehydes
this gene is the next step of hydrocarbon degradation, specially for the medium-chain alcohols, wchih derived from P450(BBa_826000).
it can coupled with P450(BBa_826000) to work.
Ray Paul Laureano-Mercado & Mark Kirollos
This actuator's purpose is to code for the output of the enzyme melamine deaminase. This enzyme eliminates an amine group from melamine to produce ammonia and ammeline and is able to remove another amine group at a rate 10 times slower than the first reaction to bring the compound to ammelide. The enzyme does not react to a measurable extent with this last compound, so it metabolizes melamine by consecutive and predictable deaminations. Its design comprises three components, which include a bicistronic ribosome binding site, the MDA gene of interest which codes from melamine deaminase, and a transcription terminator. The part needs a PoPS signal as an input in order to produce the desired enzyme and was designed to work in E. coli.
The TriA gene???s codons were modified from the original sequence and optimized for E. coli to a Codon Adaptation Index (CAI) of .76 (Puigb??, P., Guzm??n, E., Romeu, A. & Garcia-Vallv??, S. OPTIMIZER: A web server for optimizing the codon usage of DNA sequences. Nucleic Acids Research. 2007;35:W126???31.). After that, some palindromic and mRNA destabilizing sequences were removed to increase the stability of our DNA during synthesis. The sequence shown maintains a CAI of .76 and the same amino acid sequence as the original TriA gene.
The original gene coding for the protein of interest was isolated from a species of Pseudomonas (http://www.ncbi.nlm.nih.gov/nuccore/11890744). The sequences for the RBS and Terminator were sourced from BIOFAB (http://biofab.org/)
Endoglucanase gene CelC from Agrobacterium tumefaciens C58.
The source ofEndoglucanase genes (CelC) is GenBank(NC_003063).
This is Endoglucanase gene (CelC) from the genome of Agrobacterium tumefaciens C58.
sfGFP + PETase (PET-degrading enzyme, origin I. sakaiensis)
This His-tag is a sequence that allows a conjunct protein to be purified. The tag is composed of repetitions of histidines.
Conversion of the iGEM parts registry to SBOL2.1