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.
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'': <partinfo>BBa_K156012</partinfo>. 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 <partinfo>BBa_K215000</partinfo> promoter. Naturally, each gene is preceded by a standard RBS (<partinfo>B0034</partinfo>) and the transcript finishes with a strong terminator (<partinfo>B0015</partinfo>), 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.
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.
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.
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.
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).
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
Alcohol dehydrogenase from Bacillus thermoleovorans B23.
Nucleotide sequence optimized for expression in E.coli.
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.
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
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
bpul (laccase from Bacillus pumilus) with T7 promoter, RBS and HIS tag
bpul (Laccase from Bacillus pumilus) with T7, RBS and HIS tag
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.
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
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
Site directed mutagenesis to remove internal EcoRI site
tthl laccase from Thermus thermophilus with T7 promoter, RBS and His-tag
tthl (Laccase from Thermus thermophilus) with T7, RBS and HIS tag
tphB: reaction from DCD to protocatechuate
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.
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).
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.
A construction intermediate used to produce <partinfo>BBa_K338003</partinfo>. Contains the first of three genes required to synthesize polyhydroxybutyrate (PHB).
Designed to be ligated upstream of <partinfo>BBa_K338023</partinfo> and <partinfo>BBa_K338024</partinfo>.
The Registry of Standard Biological Parts
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.
bhal laccase from Bacillus halodurans with T7 promoter, RBS and His-tag
bhal laccase from Bacillus halodurans with T7 and HIS
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)
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
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
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
Long-Chain Alkane Monooxygenase of Geobacillus thermodinitrificans.
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
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
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.
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 ;
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.
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.
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.
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.
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/)
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.
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
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.
pelB + sfGFP + PETase (PET-degrading enzyme, origin I. sakaiensis)
ecol laccase from E. coli with T7 promoter, RBS and His-tag
E.coli laccase ORF with T7, RBS and HIS tag
tthl (Laccase from Thermus thermophilus) coding sequence
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.
Aldehyde dehydrogenase from Geobacillus thermoleovorans B23.
This is one of the two catalases from E. coli.
Site directed mutagensis to remove an EcoRI site.
tthl laccase from T. thermophilus with constitutive promoter J23100, RBS and His-tag
tthl laccase (T. thermophilus) with constitutive promoter and HIS tag