Coding region for Prepro-Thaumatin. Codon optimized for yeast.
Synthesized by IDT. The part is codon optimized for yeast. 3 forbidden restriction sites have been removed.
Based on: http://www.ncbi.nlm.nih.gov/nuccore/121945717
Pelargonidin producing operon - T7-B0034-F3H-B0034-DFR-B0034-eANS
Sascha Hein, Sebastian Barthel, Thomas Dohmen, Sven Jager
O-acyltransferase WSD from Acinetobacter sp. (codon usage optimized for R. opacus) as used
This biobrick codifies for the protein DGAT (Diacylglycerol O-acyltransferase) which have dual activity, it can act as a wax ester synthase or as a diacylglycerol acyltransferase. In the latter case, its function consists in mediate the final step in TGAs synthesis using a fatty acyl-CoA and a DGA. This enzyme is vital in the storage and synthesis of lipids. Based on Uniprot information, this enzyme is more likely to act as a wax ester synthase with (C12-C18)-CoA whereas with (C20)-CoA it acts as a DGAT.
Two transcriptional terminators (two biobricks BBa_B1006) were set at the end of the coding sequence. Also, to avoid RBS-CDS problems, the CDS prefix and suffix mentioned in this link (http://partsregistry.org/Assembly:RBS-CDS_issues#Standard_Assembly_and_the_RBS) were used.
The amino sequence of this part was taken from Uniprot (wax-dgaT accesion Q8GGG1; http://www.uniprot.org/uniprot/Q8GGG1) and then codon optimization for R. opacus (avoiding restriction sites for the standard assembly method) was made using the free software GeneDesigner.
lipA from B. cepacia (codon usage optimized for E. coli)
This biobrick codifies for the protein lipA from Burkholderia cepacia (Pseudomonas cepacia) which catalyzes the hydrolysis of triglycerides (Triacylglycerol + H2O = diacylglycerol + a carboxylate). In the presence of certain alcohols, it catalyzes the transesterification of TGAs to produce acyl esters and glycerol. It has only one PTM (Post-Translational Modification) which consists in a sulfide bond between 234 and 314 aa. Calcium serves as a cofactor for this enzyme (1 ion per subunit). The native signal peptide was not modified.
The sequence of a transcriptional terminator (biobrick BBa_B1006) was set at the end of the coding sequence. Also, to avoid RBS-CDS problems, the CDS prefix and suffix mentioned in this link (http://partsregistry.org/Assembly:RBS-CDS_issues#Standard_Assembly_and_the_RBS) were used (not in the sequence presented here).
The amino sequence of this part was taken from Uniprot (lipA accesion P22088; http://www.uniprot.org/uniprot/P22088) and then codon optimization for E. coli (avoiding restriction sites for the standard assembly method) was made using the free software GeneDesigner.
BBa_K1322231 is a codon-optimized biobrick part encoding the gene for alpha-santalene synthase (EC 4.2.3.82). The enzyme catalyze the conversion of the common isoprenoid intermediate farnesyl pyrophosphate (FPP) into the sesquiterpene (+)-alpha-santelene in a single step. Traces of (-)-beta-santalene and bergamontene have previously been shown to be produced by this enzyme as well. The gene, derived from a relative of the exotic sandalwood tree, has been demonstrated to produce functional terpene product in both yeast (Scalcinati et al 2012) and E. coli (data pending). This is possible due to several endogenous pathways that produce FPP as an intermediate, including the MEV and MEP pathways, In addition to being a prized fragrance, with what is often described as a warm, sweet woody scent, the sandalwood oil has been investigated for a number of other practical applications, including as a chemoprotective against carcinogenesis (Banaerjee, Ecavade, and Rao 1993) and inhibitors of viral reproduction (Koch et al 2008). References: Scalcinati et al.: Combined metabolic engineering of precursor and co-factor supply to increase α-santalene production by Saccharomyces cerevisiae. Microbial Cell Factories 2012 11:117. Banerjee, Ecavade and Rao: Modulatory influence of sandalwood oil on mouse hepatic glutathione S-transferase activity and acid soluble sulphydryl level. Cancer Letters, 68 (1993) 105 - 109 Koch et al: Inhibitory effect of essential oils against herpes simplex virus type 2. Phytomedicine 2008;15(1-2):71-8.
Two sites on the original gene sequence had to be changed by selected sense mutation codons in order to make the part RFC10 compatible.
The sequence comes from genomic DNA taken from the santalum genus of plant. It was codon optimized for expression in Saccharomyces cerevisiae. The part also contains a yeast consensus sequence before the start codon, a C-terminal strep tag, and biobrick prefixes and suffixes.
metK (from Streptomyces avermitilis, increasing avermectin production)
Erg10 encodes thiolase in Saccharomyces cerevisiae.
RgPAL1：phenylalanine ammonia lyase (PAL) coding region
The gene RgPAL1 can express Phenylalanine ammonia lyase（PAL）, which is an??enzyme??that??catalyzes??a reaction converting L-phenylalanine??to ammonia??and??trans-cinnamic acid.PAL is the first and committed step in the??phenyl propanoid pathway??and is therefore involved in the??biosynthesis??of the??polyphenol??compounds. In our project, PAL plays an important role in L-phenylalanine???s deaminization to produce trans-cinnamic acid.
The gene RgPAL1 can express Phenylalanine ammonia lyase（PAL）.
This part encodes a nematotoxic lectin from the parasol mushroom( Macrolepiota procera) under the control of pBAD promoter.
We searched the cDNA of MpL, and optimized the codon for E. coli.
B0034-CHI - Chalcone Isomerase from Petunia with strong RBS
Sascha Hein, Niklas Hummel, Sebastian Barthel, Thomas Dohmen
The Chalcone Isomerse catalyzes the reaction from Naringenin chalcone to Naringenin. This enzyme is non-essential for flavanone pathways, because the reaction takes place independently from CHI in the cytosol. The enzyme independent reaction is catalyzed by an acidic compound. Without CHI the reaction is stereo unspecific. S-Naringenin is the active compound for the most enzymes in the biochemical pathways. To prevent gaining a racemic product and losing 50% yield, CHI is used, because only the enzyme is able to form 100 % S-Naringenin. For using the CHI in our Naringenin Operon (K1497007) we added the strong RBS B0034.
Yeast expression cassette for CaMXMT1 (BBa_K801071) controlled by the yeast TEF1 promoter (BBa_K319003) and the yeast ADH1 terminator (BBa_K801012).
Composite part of BBa_K319003, BBa_K801071 and BBa_K801012
AspA is an aspartate ammonia-lyase which catalyzes the equilibrium of aspartate and fumarate + ammonium ion. With enough amount of substrate, it produces aspartate. Aspartate can be used as a chemoattractant for Escherichia coli.
tphA3: Catalyzes together with tphA2 TPA to DCD
(+)-Limonene synthase 1 with Strep-tag and yeast consensus sequence.
This part contains the (+)-Limonene Synthase 1 of ''Citrus limon''. It is preceeded by the yeast consensus sequence for improved expression and carries a C-Terminal Strep-Tag for purification or detection by westernblot. It is an improved version of BBa_I742111.
Bastian Wagner, Christian Mende, Andreas Schmidt, Max Dombrowsky
Modified rhlA coding sequence from Pseudomonas aeruginosa SQ6
RhlA together with RhlB encodes Rhamnosyltransferase I for mono-rahmnolipids biosynthesis in Pseudomonas aeruginosa.
A mutation has been done to remove the PstI restriction site contained in the coding sequence.
Yeast expression cassette for CaXMT1 (BBa_K801070) controlled by the yeast TEF2 promoter (BBa_K801010) and the yeast ADH1 terminator (BBa_K801012).
Composite part of BBa_K801010, BBa_K801070, BBa_K801012
Constitutive promoter + 37℃ induced RBS + tetR + double terminator
we took advantage of the interaction between 37?? C induced RBS, tetR gene and pTet promoter to create the temperature regulating circuit. The following is a description of our prescribed strategy. First, we use the constitutive promoter, 37?? C induced RBS (K115002), the tetR gene and terminators. We will call this part the ???Regulator???. We speculate that when the ???Regulator??? and ???Production??? sections are combined, we can achieve a high bacteria growth rate and close to no target protein producing rate at T>37??C, and low bacteria growth rate and high target protein producing rate at T<37??C.
To regulate following gene by temperature
Chalcone O-methyltransferase (OMT1) + yeast consensus sequence
Chalcone O-methyltransferase (OMT1) coding region from ''Humulus lupulus'' preceeded by yeast consensus sequence for improved expression.
This parts enables expression of pyruvate decarboxylase (pdc) and alcohol dehydrogenase B (adhB) using IPTG induction. Those enzymes are involved in conversion of pyruvate to ethanol via an acetaldehyde intermediate. The pdc and adhB sequences come from ''Zymomonas mobilis'' however, they are codon optimised for ''E. coli''.
Presence of LacZ alpha peptide upstream of the ORFs coding for the enzymes enables blue- white screening.
The Chalcone Synthase catalyzes the reaction from 4-Coumaroyl-CoA to Naringenin Chalcone. It is one of the essential enzymes in the anthocyanin pathway.
Reaction of the CHS. 4-coumaroyl-CoA reacts together with 3 Malonyl-CoA to one Naringenin Calcone. We used the CHS and verified the function in our Naringenin Operon (K1497007).
RhlI Acyl-homoserine-lactone synthase from P.aeruginosa SQ6
This sequece encodes RhlI, an acyl-homoserine-lactone synthase required for the synthesis of BHL (N-butanoyl-L-homoserine lactone), and HHL (N-hexanoyl-L-homoserine lactone) autoinducer molecules which bind to RhlR.
It includes both the complete coding sequence of RhlI and part of noncoding sequence between RhlR and RhlI gene.
It comes from the genomic sequence of P.aeruginosa SQ6.
(+)-limonene synthase 1 expression cassette for yeast
This part can be used to express Citrus limon (+)-limonene synthase 1 in yeast. The expression is controled by the yeast TEF1 promoter and the yeast TEF1 terminator.
This is a composite part consisting of the yeast TEF1 promoter (BBa_K319003), the Citrus limon (+)-limonene synthase 1 coding region (BBa_K801061) and the yeast TEF1 terminator (BBa_K801011).
This part can be used to express ''Citrus limon'' (+)-limonene synthase 1 in yeast. The expression is controled by the yeast TEF2 promoter and the yeast CYC1 terminator.
This is a composite part consisting of the yeast TEF2 promoter (BBa_K801010), the Citrus limon (+)-limonene synthase 1 coding region (BBa_K801061) and the yeast TEF1 terminator (BBa_K122003).
PCR using primers with biobrick prefix and surfix, cloned into pSB1AC3, and then eliminate Pst1 and EcoR1 using site-directed mutation.
Aromatic prenyltransferase (APT) coding region
Aromatic prenyltransferase (APT) coding region from ''Humulus lupulus'', yeast codon optimized.
Synthesized by Geneart, yeast codon optimized http://www.ncbi.nlm.nih.gov/nuccore/AB543053
xanthosine methyltransferase CaXMT1-strep
xanthosine methyltransferase CaXMT1-strep from ''Coffea arabica'' followed by a strep-tag for purification and/or detection via western blot.
http://www.ncbi.nlm.nih.gov/nuccore/AB048793 synthesized by IDT, yeast codon optimized
The UV-responsive wintergreen odor generator catalyzes the conversion of the precursor salicylic acid to the wintergreen odor methyl salicylate under extensive UV irradiation. The biosynthetic device is composed of two transcriptional devices: Rec A (SOS) promoter (BBa_J22106) and a wintergreen odor enzyme generator (BBa_J45119). The odor generator takes as input extensive UV irradiation and produce as output an enzyme that catalyzes production of an odor from a chemical precursor.
Propionate CoA transferase for P. putida
This is the DNA coding sequence for the enzyme Propionate CoA. The enzyme normally catalyzes the reaction: acetyl-CoA + propanoate <--> acetate + propanoyl-CoA. Transferase. This mutated sequence has one amino acid substitution: A243T (compared to the wild type enzyme) and is codon optimized for Pseudomonas putida in order to produce (D)-Lactyl-CoA. It extend the BBa_K1211001 biobrick from Yale 2013 and have the same purpose.
We use this enzyme as the first enzyme in the pathway to create PLA or Polylactic acid. Lactate can also act as acceptors for the enzyme. In our project, the enzyme will catalyze the reaction: acetyl-CoA + lactate <--> acetate + Lactyl-CoA.
The wild type sequence is from Clostridium propionicum genome.
Constitutive naringenin producing operon
4-coumarate--coenzyme A ligase (4CL) coding region
4-coumarate--coenzyme A ligase (4CL) coding region from ''Arabidopsis thaliana''.
Plasmid (template for PCR) provided by John A. Morgan, School of Chemical Engineering, Purdue University, West Lafayette, Indiana, USA http://www.ncbi.nlm.nih.gov/nucleotide/609339/ PMID: 15932991
Ralf Beer, Konrad Herbst, Nikolaos Ignatiadis
The IndC Indigoidine Synthetase from Photorhabdus luminescens laumondii TT01 is a nonribosomal peptide synthetase (NRPS) which produces the blue pigment indigoidine. Indigoidine itself consists of two cyclic glutamines. IndC is a single module NRPS and consists of an Adenylation-, Thiolation- and a Thioesterase-domain. The A-domain thus contains an internal Oxidation domain.
The native indC contains an internal EcoRI and SpeI cutting site which was mutated using a CPEC approach.
The coding sequence was amplified from Photorhabdus luminescens laumondii TT01 DSM15139.
With Plux gene,it can detect the concentration of AHL-LuxR protein complex and initiate the following gene Sequence and Features
Naringenin producing operon under the T7 promoter BBa_I712074
Transformed into BL21(DE3) and induced with IPTG this composite is able to produce naringenin from tyrosine. It consists of 4 genes: 4-CL (BBa_K801093) TAL (BBa_K1033000) CHI (BBa_K1497000) CHS (BBa_K1497001) For a strong and stable translation in front of each gene the RBS Bba_B0034 is implemented. For characterization the naringenin biosensor K1497020 was used. After 24 h incubation GFP fluorescence was visible to the unaided eye. We measured the relative GFP fluorescence by use of OptimaFluoroStar.
This DNA sequence codes for the enzyme CrtI, also known as phytoene desaturase. CrtI converts pytoene to lycopene which is an important step in the carotenoid biosynthesis pathway in yeast. This sequence also includes a PGK promoter and an ADH1 terminator.
While designing the sequence, we made sure that the cut sites for restriction enzymes avr2 and spe1 did not occur anywhere within the sequence.
The sequence of this part was originally taken from the genome of Xanthophyllomyces dendrorhous. This sequence can be found in the NCBI database. We then codon optimized this sequence for Saccharomyces cerevisiae using an online tool found here: http://www.encorbio.com/protocols/Codon.htm
Cytochrome A and X from Nitrosomonas europaea
(+)-Limonene synthase 1 coding region of ''Citrus limon''. Improved version of BBa_I742111.
Codes for the glucose-1-phosphate adenylyltransferase enzyme, utilized in the rate-limiting first step of e coli glycogen biosynthesis (as described in EcoCyc database). See http://BioCyc.org/ECOLI/NEW-IMAGE?type=GENE&object=EG10379 for full enzyme details.
This mutation results in greater glycogen production compared to WT.
The sequence provided here is from the CL1136 strain, with an Arg67Cys mutation.
Denitrification operon from Pseudomonas aeruginosa
Kristian Davidsen, Helen Cook and Jakob Berg Jespersen
Nitrous oxide reductase (nosZ) from Pseudomonas aeruginosa PAO1
phenylalanine ammonia lyase (PAL) coding region
Coding region for the enzyme phenylalanine ammonia lyase (PAL) from ''Rhodosporidium toruloides''
Plasmid (template for PCR) provided by John A. Morgan, School of Chemical Engineering, Purdue University, West Lafayette, Indiana, USA http://www.ncbi.nlm.nih.gov/nuccore/AX366866 PMID: 15932991
This is the CDS for TycB1 module from the Tyrocidine gene cluster of Brevibacillus Parabrevis. It is the second module in this cluster for the NonRibosomalPeptide-Synthetase of the cyclic decamer Tyrocidine. This specific module TycB1 is responsiple for the binding of the second amino acid Proline of the NRP. It consists of the C- (condensation), A- (adenylation) and the PCP- (peptide-carrier-protein) domain. After Tyc B1 follows the TycB2 module of the TycB gene which has three modules. This bio brick can be used for the design of different NRPS.
Silent mutation was found after sequencing with VR and VF2 at positions 4595 (GCC to GCG; Alanin to Alanin) and at position 4598 (CTT to CTG; Leucine to Leucine).
Tyrocidine Gene Cluster of Brevibacillus Parabrevis. Genebank File taken from http://www.ncbi.nlm.nih.gov/nuccore/af004835
luxABCDE gene ligated to the rear of T7 promoter. When transformed into BL21(DE3) E.coli strain, add different amount IPTG to reach different final concentration of IPTG in the medium can induce different level of Lux ligt emission. No substrate is needed.
luxABCDE gene ligated to the rear of T7 promoter. The original Ara and P-bad were replaced with T7 promoter, which enables its usage in our project.
T7 promoter and LuxCDABE with their RBS are from Partsregistry.
LuxCDABEG with corresponding six RBS(from K325909)
predicted glycogen synthesis protein (w/ UTR&term)
Codes for a protein involved in the biosynthesis of glycogen. Overproduction stimulates glycogen synthesis. Influences the first two enzymes in the e coli glycogen biosynthesis pathway (glucose-1-phosphate adenylyltransferase, glycogen synthase). See http://BioCyc.org/ECOLI/NEW-IMAGE?type=GENE-IN-REG-SUMMARY&object=EG11381 for more information.
Genomic sequence for glgS as provided on EcoCyc database.
This is the CDS for TycA_deltaCom module from the Tyrocidine gene cluster of Brevibacillus Parabrevis. It is the first module in this cluster for the NonRibosomalPeptide-Synthetase of the cyclic decamer Tyrocidine. This specific module Tyca_deltaCom is responsiple for the binding of the first amino acid D-Phenyl of the NRP. It consists of the A- (adenylation), PCP- (peptide carrier protein) and the E- (epimerization) domain. After Tyc A follows the TycB gene with three modules. This bio brick can be used for the design of different NRPS. It was used in the synthetic Tripeptide I of our project.
This is just the coding sequence. We sequenced with VR and VF2 primer, not mutations were found.
This is the coding region for the Sex-Lethal Gene from which can be used to control splicing events.
This was part was made using PCR from a construct synthesized by Mr. Gene. It intons have been artificially removed.
Riboflavin synthesis gene cluster from shewanella oneidensis
This gene cluster consists of four different genes that form a single operon.
we eliminated three illegal restriction sites.
B-carotene monooxygenase, Produces retinal from B-carotene. ninaB gene from Drosophila.
This coding region encodes a beta-carotene deoxygenase, that splits beta-carotene into two retinal molecules.
No considerations have been made so far concerning design
RBS + PhaC (sp. MBEL 6-19) + RBS + PCT (CP)
7-methylxanthine N-methyltransferase CaMXMT1-strep
7-methylxanthine N-methyltransferase CaMXMT1 from ''Coffea arabica'' followed by a strep-tag for purification and/or detection via western blot.
Synthesized by IDT, Yeast codon optimized
This part codes for the fusion of Pyruvate decarboxylase and Alcohol dehydrogenase from ''Zymomonas mobilis''. Fused enzymes increase ethanol yields and enable its faster production.
The part is based on the BBa_K173003 - the enzymatic components of this BioBrick were fused using MABEL technology. The fusion removed start codon in adhB, stop codon in pdc and the RBS inbetween.
''Z. mobilis'' gDNA codon optimised for ''E. coli''
The Chalcone Isomerse catalyzes the reaction from Naringenin chalcone to Naringenin. This enzyme is non-essential for flavanone pathways, because the reaction takes place independently from CHI in the cytosol. The enzyme independent reaction is catalyzed by an acidic compound. Without CHI the reaction is stereo unspecific. S-Naringenin is the active compound for the most enzymes in the biochemical pathways. To prevent gaining a racemic product and losing 50% yield, CHI is used, because only the enzyme is able to form 100 % S-Naringenin. Reaction of the CHI. A proton shift of naringenin chalcone builds naringenin. We used the CHI and verified the function in our Naringenin Operon (K1497007).
Nitrite Reductase (nirS) from Pseudomonas aeruginosa PAO1
This gene codes for the nitrite reductase (nirS) that converts nitrite (NO2-) into nitric oxide (NO). It is cloned into the pSB1C3
Nitric oxide reductase (norC) from Pseudomonas aeruginosa PAO1
Yeast expression cassette for CaDXMT1 (BBa_K801072) controlled by the yeast TEF2 promoter (BBa_K801010) and the yeast ADH1 terminator (BBa_K801012)
Composite part of BBa_K801010, BBa_K801072 and BBa_K801012
Combination of oprF and rib-synth coding sequence under control of T7 promoter
Codes for the glycogen synthase enzyme, utilized in the second step of e coli glycogen biosynthesis (as described in EcoCyc database). See http://biocyc.org/ECOLI/new-image?type=GENE&object=EG10377 for full enzyme details.
Comes from standard genomic sequence (glgA) for the e coli glycogen synthase enzyme.
gluc1phos adenylyltrans (with 5'UTR & term)
The sequence provided here is from the CL1136 strain, with an Arg67Cys mutation in the glgC gene.
Pelargonidin producing operon (low yield)- T7-B0034-F3H-B0034-DFR-B0034-ANS
Sascha Hein, Sebastian Barthel, Thomas Dohmen
Juliette Paillet, Alexandre Duprey, Emiel van der Kouwe
B0034-CHS-B0034-CHI - Composite part of RBS-CHS and RBS-CHI
This device is able to generate Naringenin from 4-courmaroyl-CoA, if put under a promotor. This device is the second composite part for the assembly of our Naringenin Operon (K1497007). Part 1: K1497004: B0034-CHS - Chalcone Synthase from Gerbera with strong RBS The Chalcone Synthase catalyzes the reaction from 4-Coumaroyl-CoA to Naringenin Chalcone. It is one of the essential enzymes in the anthocyanin pathway. For using the CHI in our Naringenin Operon (K1497007) we added the strong RBS B0034. Part 2: K1497003: B0034-CHI - Chalcone Isomerase from Petunia with strong RBS The Chalcone Isomerse catalyzes the reaction from Naringenin chalcone to Naringenin. This enzyme is non-essential for flavanone pathways, because the reaction takes place independently from CHI in the cytosol. The enzyme independent reaction is catalyzed by an acidic compound. Without CHI the reaction is stereo unspecific. S-Naringenin is the active compound for the most enzymes in the biochemical pathways. To prevent gaining a racemic product and losing 50% yield, CHI is used, because only the enzyme is able to form 100 % S-Naringenin. For using the CHI in our Naringenin Operon (K1497007) we added the strong RBS B0034.
This part can be used to express Citrus limon (+)-limonene synthase 1 in yeast. The expression is controled by the TEF1 promoter and the CYC1 terminator.
This is a composite part consisting of the yeast TEF1 promoter (BBa_K319003), the ''Citrus limon'' (+)-limonene synthase 1 coding region (BBa_K801061) and the yeast CYC1 terminator (BBa_K122003).
Genomic sequence glgS as provided on EcoCyc database.
Riboflavin synthesis gene cluster from s. oneidensis under control of a strong Anderson promoter
In enzymology, a benzoylformate decarboxylase (EC 4.1.1.7) is an enzyme that catalyzes the following chemical reaction: benzoylformate + H+ ⇌ benzaldehyde + CO2 However, in our project we get an engineered benzoylformate decarboxylase, which could catalyze formaldehyde into Glycolaldehyde and Dihydroxyacetone, from CAS. If you use it to catalyze reaction in vivo, It needs one cofactor, thiamin diphosphate.
Benzoylformate decarboxylase is flanked by prefix and suffix provided by iGEM.
Benzoylformate decarboxylase(BFD) comes from Pseudomonas putida, which could cleave carbon-carbon bonds.
This DNA sequence codes for the binary enzyme CrtYB. CrtY (Lycopene Beta-cyclase) converts lycopene into beta-carotene and serves as the last step in beta-carotene biosynthesis in Saccharomyces cerevisiae. CrtB is acts further upstream of CrtY in the pathway and is called Phytoene Synthase. CrtB converts geranylgeranyl diphosphate to phytoene.
encodes a nematotoxic lectin from the parasol mushroom(Macrolepiota procera) under the control of a constitutive promoter.
We optimized the codon sequence for E. coli. and add a constitutive promoter J23100 upstream the coding sequence of rMpL, so that this parts can express rMpL lectin continuously.
It comes from Macrolepiota procera cDNA.
RcsB is a receiver protein in a two component phosphorelay system. Up-regulation of RcsB has been shown to induce colanic acid production. This is via the activation of the ugd operon which is required for capsule synthesis.
RcsB also upregulates ftsZ which increases the rate of cell division.
This biobrick codifies for the protein lipA from Burkholderia cepacia (Pseudomonas cepacia) which catalyzes the hydrolysis of triglycerides (Triacylglycerol + H2O = diacylglycerol + a carboxylate). In the presence of certain alcohols, it catalyzes the transesterification of TGAs to produce acyl esters and glycerol. It has only one PTM (Post-Translational Modification) which consists in a sulfide bond between 234 and 314 aa. Calcium serves as a cofactor for this enzyme (1 ion per subunit).
The sequence of a transcriptional terminator (biobrick BBa_B1006) was set at the end of the coding sequence. Also, to avoid RBS-CDS problems, the CDS prefix and suffix mentioned in this link (http://partsregistry.org/Assembly:RBS-CDS_issues#Standard_Assembly_and_the_RBS) were used.
The amino sequence of this part was taken from Uniprot (lipA accesion P22088; http://www.uniprot.org/uniprot/P22088) and then codon optimization (avoiding restriction sites for the standard assembly method) was made using the free software GeneDesigner.
p70-CsgA-His*2, double His-tagged curli generator
Expresses the glgC, glgA, and glgS proteins/enzymes usually not expressed in e. coli.
Using different RBSs so that secondary structures and self pairing don't occur.
This composite part is the fusion device of the Biobrick parts K1033001 by Uppsala 2013 and K1033000 by. It is able to produce 4-coumaroyl-CoA from tyrosine, if put under a promotor. This device is the first composite part for the assembly of our Naringenin Operon (K1497007). We used the previously announced Biobricks to assemble this part and verified the function in our Naringenin Operon (K1497007).
Modified rhlB coding sequence from Pseudomonas aeruginosa SQ6
RhlB together with RhlA encodes Rhamnosyltransferase I for mono-rahmnolipids biosynthesis in Pseudomonas aeruginosa. This part includes bp upstream noncoding gene and bp downstreanm gene of RhlB. Two mutations have been done to remove the PstI restriction sites contained in the coding sequence, which won't change the amino acid sequence.
Two mutations have been done to remove the PstI restriction sites contained in the coding sequence, which won't change the amino acid sequence.
Prepro-Thaumatin expression cassette for yeast
This part can be used to constitutively express Prepro-Thaumatin in ''S. cerevisiae'' under the control of the TEF1-promoter and the TEF1-terminator.
Based on part BBa_K801080 (Prepro-Thaumatin).
Re-designing "The Biosurfactator" The first Central American BioBrick
Pseudomonas aeruginosa species of bacteria produce the natural rhamnosyltransferase gene complex (RhlAB). This is the key enzyme responsible for transferring the rhamnose moiety to the b-hydroxyalkanoic acid moiety to biosynthesize rhamnolipid, which is a biomolecule with surfactant properties, but the natural RhlAB gene has illegal restriction sites that make it incompatible with the Assembly Standard Protocol 10, specifically the Pstl1 restriction sites. The design and construction of the rhamnosyltransferase gene into a BioBrick part was the main goal and achievement of the iGEM Panama Team 2010, but since the beginning of this year???s iGEM project we failed in producing the rhamnolipid compound into our E. coli based-factory, because our last year???s BioBrick appeared to have been denaturalized. We then ordered our BioBrick part BBa_K424018 (iGEM Panama team 2010) from The Registry and once it arrived we ran some tests and we noticed that the part was no longer fitted into the plasmid backbone. As scientist and iGEMers, we have the responsibility to deliver a functional BioBrick, thus according to iGEM???s competition new slogan, ???Quality not Quantity???, we have embraced the challenge of re-designing and re-building the same BioBrick part from last year in order to comply this new standard based on quality. How to use it as a BioBrick? : We developed this standarized part to be inserted into E. coli for rhamnolipid production through the mutation of the gene that is responsible to synthesize the key enzyme rhamnosyltranferase (RhlAB) to be compatible with Assembly Standard 10. We have achieved this by performing several silent mutations using the QuikLighting Multi Site-Directed Mutagenesis Kit from Stratagene. This rhamnosyltransferase BioBrick (Rh1AB_BB) is ready to be tested on an expression plasmid device following the Assembly Standard Protocol 10.
The RhlAB gene complex has shown illegal restriction sites according to the assembly standard protocol 10. We faced this problem by applying the mutagenesis protocol that consist in doing silent mutations that change the nucleotides of our rhlAB gene that are cut by the Pstl restriction enzyme. This will give us a standardized coding sequence of the rhlAB gene compatible with the Assembly Standard protocol 10 and a new BioBrick part for the Registry.
Pseudomonas aeruginosa from INDICASAT lab. and the genomic sequence of RhlAB was reviwed in genBank (ID 878955 and 878954) http://www.ncbi.nlm.nih.gov/nuccore/L28170.1
This composite part to produce naringenin from tyrosine, if put under a promotor. It consists of 4 genes: 4-CL (BBa_K801093) TAL (BBa_K1033000) CHI (BBa_K1497100) CHS (Bba_K1497101) For a strong and stable translation in front of each gene the RBS Bba_B0034 is implemented.
The functionality of this construct is verified by putting the device under a promotor. Proofing functionality leaded to Bba_K1497008 (Naringenin Operon under the constitutive promotor J23100) and Bba_K1497008 (Naringenin Operon under the T7 Promotor).
Converts the amino acid tryptophan into 5-HTP, which is a precursor to serotonin. Soon to be edit
Optimised for E.coli, removed illegal restriction enzyme sites
Test plataform for rhamnosyltransferase BioBrick (Rh1AB_BB) expression in E. coli
PCR using primers with biobrick prefix and surfix, cloned into pSB1AC3, and then eliminate Pst1 using site-directed mutation.
pelB-EstCS2-his expression (GFP reporter)
This DNA sequence codes for the enzyme CrtE, also known as geranylgeranyl diphosphate synthase. CrtE serves as the first step in carotenoid biosynthesis in Saccharomyces cerevisiae by converting farnesyl diphosphate (which is naturally found in yeast) to geranylgeranyl diphosphate.
Pelargonidin producing operon B0034-F3H-B0034-DFR-B0034-eANS
phenylalanine ammonia lyase (PAL) + yeast consensus sequence
phenylalanine ammonia lyase (PAL) coding region from ''Rhodosporidium toruloides'' preceeded by the yeast consensus sequence for improved expression.
Plasmid (template for PCR) provided by John A. Morgan, School of Chemical Engineering, Purdue University, West Lafayette, Indiana, USA http://www.ncbi.nlm.nih.gov/nuccore/AX366866 PMID: 1593299
Modified rhlAB coding sequence from Pseudomonas aeruginosa SQ6
Modified rhlABRI operon genes from Pseudomonas aeruginosa SQ6
Expresses glgC mutant gene (coding for the glucose-1-phosphate adenylyltransferase) and glgA gene (coding for glycogen synthase).
Composite of genomic sequences coding for proteins involved in e. coli glycogen biosynthesis.
Expresses glgC mutant gene (coding for the glucose-1-phosphate adenylyltransferase) and glgS gene (predicted glycogen synthesis protein). This part is greatly reduced in size from the ideal glycogen synthesis actuator by the removal of glgA.
This DNA sequence codes for the enzyme CCD1, also known as carotenoid cleavage dioxygenase 1. CCD1 cleaves beta-caroten to form two molecules of beta-ionone. This enzyme completes the beta-ionone biosynthesis pathway in yeast. This sequence also includes a PGK promoter and an ADH1 terminator.
Diguanylate cyclases produce cyclic diGMP (c-di-GMP), a secondary messenger. Diguanylate Cyclase (DgcZ), also called YdeH, is present in Escherichia coli and the stimulus that controls it is Zinc.
The sequence did not have any restriction sites to remove so it was immediately PCRed out of the genome and had the registry prefix and suffix added to it. It was then ligated into the pSB1C3 vector. YdeH however, is toxic to E. coli cells.
The source of this part is Escherichia coli MC40100 genomic DNA which was provided by the Tracy Palmer lab in the Molecular Microbiology department of the University of Dundee, UK.
Pseudomonas sp. MBEL 6-19 PHA synthase 1
frr (from Streptomyces avermitilis, increasing avermectin production)
Frr gene encodes the ribosome recycling factor (RRF), which is involved in the release of ribosomes from the translational post-termination complex for a new round of initiation. In comparison to the wild-type strain, avermectin yield was increased significantly by 3- to 3.7-fold in transformants 31267(pFRR-1139) and 31267(pFRRerm-1139), both of which contained multiple frr copies, compared with that in the control strains. The avermectin productivity of each culture was quantitatively measured by HPLC analysis. Research indicated that frr overexpression promoted cell growth as well as the expression of ave genes (including pathway-specific positive regulatory gene aveR for avermectin biosynthesis and ave structural genes), leading in turn to avermectin overproduction. Different from S-adenosylmethionine synthetase gene (metK), frr gene revealed a ??????copy number effect??????: multiple copies of frr had a greater promoting effect on avermectin production than a single copy in each of the three transformed S. avermitilis strains.
The metK gene was amplified by PCR with genomic DNA of S. avermitilis(具体型号) as template using primers
The Beta-carotene 15'15'-monooxygenase generator from the D. melanogaster ninaB gene, cleaves beta-carotene into two retinal molecules. Used together with BBa_274210 (beta-carotene generator) it should induce retinal biosynthesis in e. coli.
A constitutive sigma-70 promotor was chosen. Terminator is lacking on this part.
[link:https://dgrc.cgb.indiana.edu/product/View?product=4630 Drosophila genomics ressource centre], Indiana University. Found via [link:http://www.flybase.org flybase], ID: [link:http://flybase.org/reports/FBcl0129473.html FBcl0129473]
p70-CsgA-His, His-tagged curli generator
Ammonium monooxygenase from Nitrosomonas europaea
Nitric oxide reductase (norB) from Pseudomonas aeruginosa PAO1
GldA - glycerol dehydrogenase from Escherichia coli
The Gene gldA encodes glycerol dehydrogenase from Escherichicha coli. The overexpression of glycerol dehydrogenase in E. coli can be used as a way for endogenous mediator production. Many derivates of glycerol dehydrogenase are small, water-soluble redoxmolecules, which have mediator properties. In enzymology, a glycerol dehydrogenase (EC 1.1.1.6) is an enzyme belonging to the family of oxireductases that catalyzes the chemical reaction: Glycerol + NAD --> Glycerone + NADH + H
Riboflavin synthesis gene cluster from s. oneidensis under control of a medium Anderson promoter
Naringenin - chalcone synthase (CHS) coding region
Naringenin - chalcone synthase (CHS) coding region from ''Hypericum androsaemum''
Plasmid (template for PCR) provided by John A. Morgan, School of Chemical Engineering, Purdue University, West Lafayette, Indiana, USA http://www.ncbi.nlm.nih.gov/nucleotide/11096318/ PMID: 15932991
This is the coding sequence for a Polyhydroxyalkanoate (PHA) synthase 1 from Pseudomonas resinovorans. The enzyme takes the monomer (D)-lactyl-CoA and creates the PLA homopolymer. This sequence is codon optimized for pseudomonas putida it includes four mutations which were found to increase yields of PLA (Yang et al.) site-directed mutagenesis, the mutation leads to production of high molecular weights of P(3HB-co-LA); site-directed mutagenesis, the type II PHA synthase 1 is engineered to accept short-chain-length hydroxyacyl-CoAs including lactyl-CoA and 3-hydroxybutyryl-CoA as substrates and support the synthesis of P(3HB-co-LA) by site-directed mutagenesis of four sites, i.e. E130, S325, S477, and Q481 We note that unlike BioBrick BBa K1211002 Yale 2013, one of the mutation is not on the Serine 477 on Serine 475. which is able to do the reaction: lactylCoA + PLA(n) = PLA(n+1).
We used this sequence as second enzyme in order to produce PLA. In fact it will take lactyl coA as substract and polymerise it into PLA homopolymere
O-acyltransferase WSD from Acinetobacter sp. (codon usage optimized for R. opacus)
Two transcriptional terminators (two biobricks BBa_B1006) were set at the end of the coding sequence. Also, to avoid RBS-CDS problems, the CDS prefix and suffix mentioned in this link (http://partsregistry.org/Assembly:RBS-CDS_issues#Standard_Assembly_and_the_RBS) were used (as in the rest of biobricks, the prefix and suffix are not present in the sequence).
Naringenin - chalcone synthase (CHS) + yeast consensus sequence
Naringenin - chalcone synthase(CHS) coding sequence from ''Hypericum androsaemum'' preceeded by yeast consensus sequence for improved expression.
Using parts BBa_K646000 and BBa_I719005, a T7 promoted Hemeoxygenase was created by digesting the T7 biobrick with S and P and ligating it to the HO insert also digested with S and P. Successful expression should result in green colonies due to the presence of biliverdin.
Riboflavin synthesis gene cluster from s. oneidensis under control of T7 promoter and strong RBS
Crystal protein gene (cry11Aa) from Bacillus thuringiensis subsp. Israelensis.
The cry weapon system produce crystal protein, targetting the wrigglers, larvae of mosquitoes. The cry11Aa gene is cloned from Bacillus thuringiensis subsp. Israelensis. The cry protein is controlled by the tetR-repressible promoter PtetR (BBa_R0040), which in turn is regulated by a temperature control system. When E.coli is released into environment (<37C), tetR begins to degrade, resulting in the promoter PtetR (BBa_R0011) expressing downstream genes, cry11Aa (BBa_K332012) and Green Fluorescent Protein (GFP)(BBa_E0040).
Procedures (I) Culture Bti. Bacillus thuringiensis subsp. Israelensis (BCRC15860) 1. Prepare agar plate for Bti. Beef extract 3.0g Peptone 5.0g Agar 15.0g ddH2O 1 L *adjust PH to 7.0 *No antibiotic *Be aware of contamination 2. Plate Bti. on the medium and incubate the cultures at 30??C overnight. (II) Clone cry11Aa from Bti. into TA vector 1. Extract genomic DNA of Bti. by liquid nitrogen. 2. Add some ddH2O to dilute the DNA. 3. Design primers Vf2: ATTCAATAAAAGGTGGAATGAATTATGGA Tm: 53??C VR: GTGCTAACATGACTTCTACTTTAGT Tm: 52.8??C 4. Find the best PCR condition by gradient PCR. Anneling temperature: 51??C??10??C 5. PCR by B-taq plus on the best condition Template DNA（10ng/μl） 2.0 B-taq buffer 5.0 dNTP（2mM） 5.0 forward primer（10μM） 1.5 reverse primer（10μM） 1.5 B-taq plus DNA polymerase（2Kb） 1.0 ddH2O 34.0 Total 50 μl 6. Digestion to confirm the cry11Aa fragment and enzyme sites. 7. TA clone 8. DNA sequencing (III) PCR mutagenesis at two enzyme sites --- EcoR1 and Spe1 1. Design primers by primerX. EcoR1-Vf2: CGG GTA CAA TCT CAG AAC TCG GGA AAT AAT AGA A EcoR1-VR: TTC TAT TAT TTC CCG AGT TCT GAG ATT GTA CCC G Spe1-Vf2: ATA ATG AAT GGG GAG GAC TGG TTT ATA AGT TAT TAA TGG G Spe1-VR: CTT CCC CCA TTA ATA ACT TAT AAA CTA GTC CTC CCC ATT CAT 2. Digestion to confirm the fragments (IV) PCR construction of Biobrick parts 1. Design primers by Assembly standard 10. Vf2: GTTTCTTC GAATTC GCGGCCGC T TCTAG ATGGAAGATAGTTCTTTAGATACT VR: GTTTCTTC CTGCAG CGGCCGC T ACTAGT A CTACTTTAGTAACGGATT 2. PCR condition 3. Ligation to backbones(Psb1C3). (V) Transform into E.coli 1. Thaw competent cells and BBa_K332012 plasmid on ice. 2. Add 2ul plasmid to competent cell and place in ice for 5 minutes. 3. Put the transformed cells into 42℃ water bath for 45 seconds. 4. Plate the cells on the appropriate media and antibiotic, such as agar plates with 25 ??g/ml kanamycin. 5. Incubate the cultures at 37??C overnight. (VI) Culture with mosquito larvae and observe
Crystal protein gene (cry11Aa) from Bacillus thuringiensis subsp. Israelensis. BCRC Number: 15860 Organism: Bacillus thuringiensis subsp. israelensis de Barjac History: << ATCC << USDA << ONR 60 << H. de Barjac << L. J. Goldberg 60 Other Collection No.: ATCC 35646 Source: sewage, Israel Characterization: Production of delta-endotoxin against dipteran larvae --B3009 Others: referred to as the type strain --B3008 Growth Conditions: 30??C , Medium ID: 3 Oxygen Requirement: Aerobic Biosafety Level: 1 References: Temeyer, K. B. 1984. Larvicidal activity of Bacillus thuringiensis subsp. israelensis in the dipteran Haematobia irritans. Appl. Environ. Microbiol.47 : 952-955. PMID: 6742837. Demezas, D. H. and J. Bell. 1995. Evaluation of low molecular weight RNA profiles and ribotyping to differentiate some Bacillus species. Syst. Appl. Microbiol.18 : 582-589.
4-coumarate - coenzym A ligase (4CL) + yeast consensus sequence
4-coumarate - coenzym A ligase (4CL) coding region from ''Arabidopsis thaliana'' preceeded by the yeast consensus sequence for improved expression.
Expresses B-carotene monooxygenase on a constitutive promotor
This generator expresses a beta-carotene 15,15'-monooxygenase,on a constitutive promotor, that catalyses retinal production from beta-carotene via the following reaction: beta-carotene + O2 = 2 retinal
The brick requires B-carotene supply to function. This can either be supplied externally, or alternatively the BBa_K274210 B-carotene generator will induce full B-carotene synthesis in E. coli.
The ninaB gene was originally cloned from drosophila melanogaster, and cDNA was aquired for production of the biobrick from the Drosophila Genome Ressource Center. Promotor+rbs brick BBa_J13002 and terminator BBa_B0015 were used.
B0034-eANS (engineered anthocyanidin synthase with strong RBS)
Sascha Hein, Sven Jager, Thomas Dohmen, Sven Rumpf
Gene was E. coli optimated synthezied by Eurofins.
tphB: reaction from DCD to protocatechuate
O-methyltransferase 1 (OMT1) coding region
O-methyltransferase 1 (OMT1) coding region from ''Humulus lupulus''
Constitutive promoter+37℃ induced RBS+tetR+double terminator+Ptet+RBS+GFP+double terminator
We want to utilize a temperature-regulated mechanism to control the expression period of a crystal protein. Therefore, we took advantage of the interaction between 37?? C induced RBS, tetR gene and pTet promoter to create the temperature regulating circuit. The following is a description of our prescribed strategy. First, we use the constitutive promoter, 37?? C induced RBS (K115002), the tetR gene and terminators. We will call this part the ???Regulator???.
Synechocystis hemeoxygenase with a Ribosome Binding Site (RBS), oxidises Heme into biliverdin which absorbs light in the 360 nm and 650 nm wavelength producing a green colouration.
The gene was digested with X and S before ligation, several ligations were done to ensure correct orientation.
B0034-CHS - Chalcone Synthase from Gerbera with strong RBS
The Chalcone Synthase catalyzes the reaction from 4-Coumaroyl-CoA to Naringenin Chalcone. It is one of the essential enzymes in the anthocyanin pathway. For using the CHI in our Naringenin Operon (K1497007) we added the strong RBS B0034.
PCR using primers with biobrick prefix and surfix, cloned into pSB1AC3, and then eliminate Pst1 and Not1 using site-directed mutation.
3,7-dimethylxanthine N-methyltransferase CaDXMT1-strep
3,7-dimethylxanthine N-methyltransferase CaDXMT1 from ''Coffea arabica'' followed by a strep-tag for purification and/or detection via western blot.
http://www.ncbi.nlm.nih.gov/nuccore/AB084125 synthesized by IDT, yeast codon optimized
RhlR regulatory protein from P. aeruginosa SQ6
Coding a transcription regulator RhlR. RhlR is necessary for transcriptional activation of the rhlAB genes encoding the rhamnosyltransferase. It also functions as a transcriptional activator of elastase structural gene (lasB). RhlR binds to autoinducer molecules BHL (N-butanoyl-L-homoserine lactone), and HHL (N-hexanoyl-L-homoserine lactone). This part includes the noncoding sequence of RhlR.
It includes both the complete coding sequence and part of noncoding sequence of RhlR.
It comes from the genomic sequence of Pseudomonas aeruginosa SQ6.