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.
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
CheB chemotaxis coding sequence (protein glutamate methylesterase)
CheB is part of the chemotaxis pathway in E. coli.
Checked (DE, VC, KN). Part ready for synthesis. 8/3/4
NCBI - E. Coli strain K12 substrain MG1655
phycocyanobilin:ferredoxin oxidoreductase (PcyA) from synechocystis
Second of two required phycocyanobilin biosynthetic genes.
1-aminocyclopropane-1-carboxylate (ACC) synthase
This is the protein coding sequence of 1-aminocyclopropane-1-carboxylate synthase, which catalyses the formation of 1-aminocyclopropane-1-carboxylate and methylthioadenosine from S-adenosyl methionine (AdoMet). (S-adenosyl-L-methionine <=> 1-aminocyclopropane-1-carboxylate + methylthioadenosine). This enzyme is commonly referred to as ACC synthase.
Removal of the C-terminal amino acids. According to White, M. F., Vasquez, J., Yang, S. F., & Kirsch, J. F. (1994). PNAS, 91(26), 12428-32. (http://www.ncbi.nlm.nih.gov/pubmed/7809054) - this truncation has been reported to have no effect on specific activity.
Malus domestica (Apple), codon optimized for E. coli, with the C-terminal amino acids -YYNVPEVNGGSQSSHLSHSRRQSLTKWVSRLSFDDRGPIPGR* removed
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.
tthl (Laccase from Thermus thermophilus) coding sequence
Strong RBS (BBa_B0030). Ho1 coding region (BBa_K566022). ho1, along with pcyA, converts heme into the chromophore phycocyanobillin (PCB).
Ho1 comes from cyanobacteria cyanobacterium Synechocystis.
yhjH under the control of constitutive promoter J23112
The product of this gene is a phosphodiesterase which is involved in regulating the levels of c-di-GMP, a bacterial signaling molecule. High levels of c-di-GMP promote biofilm synthesis and inhibit flagella by binding to YcgR which then acts as a "brake" on FliG and FliM. Levels of c-di-GMP are elevated and motility is reduced in ''yhjH''-mutants. Here we used 3 different constitutive [http://partsregistry.org/Part:BBa_J23100 Anderson promoters] from the 2006 Berkeley group to test how different levels of constitutive ''yhjH'' expression affect the motility of ''E. coli''.
* ''yhjH'' was amplified from genomic DNA of ''E. coli'' DH10B via PCR using the following primers: ** Fwd: 5'-gtttcttcgaattcgcggccgcttctagatgataaggcaggttatccagc-3' ** Rev: 5'-gtttcttcctgcagcggccgctactagtattatagcgccagaaccgccgtattcagc-3'
* ''yhjH'' was amplified from genomic DNA of ''E. coli'' str. K-12 substr. DH10B, complete genome (CP000948.1). * J23100 information was taken from the partsregistry and physical DNA from the 2012 distribution kit.
This sequence codifies an enzyme, Cholesterol Oxidase. This enzyme transforms the 3-OH group from the cholesterol into 3-keto; this reaction is not directly related with the elimination of the oxidation in the 7 position of the 7-ketocholesterol molecule. However, it is an "activation" reaction that unleashes the following reactions of the synthetic metabolic pathway, this occurs at the beginning of the degradation of sterols of many microbial pathways. The first step of this reactions is the oxidation of 3B-OH group, catalyzed by extracellular cholesterol oxidase.
This part was synthesized, this originally belongs to Rhodococcus jostii RHA1 genome.
pck codes for Phosphoenolpyruvate carboxykinase (Pck), a kinase involved in gluconeogenesis in E. coli
No codons were modified to make this part
This is a modified I746370 sensitivity tuner. Instead of a GFP reporter, this one has a firefly luciferase (from Promega's pGL4.10luc2 vector) as one reporter gene. The arabinose promoter has been removed from the original part so now it is possible to assemble this part behind any promoter to get an enhanced luciferase signal.
arabinose promoter removed, GFP exchanged with luciferase compared to the original part I746370
* luciferase from pGL4.10luc2 (Promega) * the rest from the partsregistry
Human guanylate cyclase1,soluble, beta 3 unit
Soluble guanylate cyclases are heterodimeric proteins that catalyze the conversion of GTP to 3',5'-cyclic GMP and pyrophosphate. It is important for smooth muscle relaxation in the cardiovascular system. sGC contains an HNOX domain, which serves as a receptor for ligands such as nitric oxide, oxygen and nitrovasodilator drugs.There are several different isoforms of sGC subunit ,but the cardiovascular system abundant with alpha 3 unit and beta 3 unit.
We used lentiviral vector to transfected alpha 3 unit and beta 3 unit to HEK293 cells together. Since soluble guanylate cyclases (sGC) are heterodimeric proteins that catalyze the conversion of GTP to 3',5'-cyclic GMP(cGMP) and pyrophosphate. The level of cGMP will be up regulated.The positive control was HEK293 cells that treat with Sodium Nitroprusside ,a NO donator that activate sGC and up regulate the level of cGMP. A negative control was made by transfecting an empty vector that does not contain sGC subunit. We used Elisa to detect cGMP level.
We get the informations from this link: http://www.ncbi.nlm.nih.gov/gene/2983
dnMyD88-linker-Rluc-linker-PEST191 is a part where dnMyD88 (very important protein in signaling cascade through TLR4 receptor) is linked to Rluc (reporter) and PEST191 (degradation tag). Both linkers are made of six aminoacids.
dnMyD88-linker-Rluc-linker-PEST191 was made by using PCR ligation technique. It was difficult since we had some problems with primers. This part is cloned in BioBrick vector pSB1AK3.
A luciferase from Promega's pGL4.10luc2 vector.
sequence of this vector is already published by Promega
linker fused to omega fragment of TEM beta-lactamase
Lux pI+RBS+LuxR+2TM+Lux pR+RBS+LuxI+2TM+Lux pR+RBS+GFP+2TM+Lux pR+RBS+Ndh+2TM
This is the protein coding sequence of S-adenosyl methionine synthetase, which catalyses the formation of S-adenosyl methionine synthetase (AdoMet) from ATP and methionine (ATP + L-methionine + H(2)O <=> phosphate + diphosphate + S-adenosyl-L-methionine). Other names for this enzyme are AdoMet synthetase, methionine adenosyltransferase. It is the product of the metK gene in E. coli.
The genomic sequence corresponding to this part does not contain any illegal restriction sites with respect to RFC10, so no mutagenesis of the native sequence was required.
Part was cloned from genomic DNA of Escherichia coli strain BL21(DE3) using PCR.
This part codes ChiA1 and its RBS. Its start codon is not AUG but GUG.
This composite part, designed and submitted by the 2016 Broad Run iGEM Team, contains an gene from the fungus S.ferax that codes for an alpha amylase enzyme. This composite part contains a constitutive promoter, coding sequence, kozak sequence, a mating factor secretion tag, and a terminator. The amylase enzyme can be expressed by cloning this part into a plasmid without a promoter built in (such as the S.cerevisiae vector pRS426).
Codon optimized for S.cerevisiae, EcoR1 and Spe1 sites removed.
The coding sequence of this part comes from the fungus S.ferax
This is the part we actually created to test the glue production by E. coli. HfsG and HfsH come from Caulobacter crescentu. Caulobacter crescentus is an aquatic, Gram-negative bacterium that divides asymmetrically and is able to synthetize a strong glue. This glue is mainly made of a polysaccharide. There are different proteins needed to synthetize, export and attach it to the stalk of Caulobacter. To see the hole system, please see our wiki page [1]. In our project, we would like this glue to be produced by Escherichia coli. As E. coli does have homolog genes for many of these proteins, but not for HfsG and HfsH, we decided to create a plasmid including only the genes coding for these two proteins. HfsG is a glycosyltransferase and HfsH is a carbohydrate esterase. RFP has been added as a reporter.
As many mutations were needed to make the part compatible with the standard 10, we decided to make hfsG and hfsH synthetized by GeneArt [2]. We also optimized it for E. coli. We mean that the codons were changed to favour the most present in E. coli.
HfsG and HfsH sequences come from Caulobacter crescentus.
DhaA31 TCP(trichlorophenol)->2,3-DCP(2,3-dichloropropano)
The enzyme DhaA is of particular interest because it enables a very slow conversion of the toxic artificial compound and suspected human carcinogen 1,2,3-trichloropropane (TCP) to 2,3-dichloropropane-1-ol under laboratory conditions .And DhaA31 is a mutant of DhaA ,DhaA31 has large aromatic residues at two of the three randomized positions and at two positions modified by site-directed mutagenesis. These changes enhance activity towards TCP by decreasing the accessibility of the active site to water molecules, thus promoting the formation of an activated complex.
Ray Paul Laureano-Mercado & Mark Kirollos
This part is the coding sequence for the enzyme melamine deaminase, optimized for usage in E. coli. 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.
The TriA gene???s codons were modified from the original and optimized for E. coli to a Codon Adaptation Index (CAI) of .76. After that, some palindromic and mRNA destabilizing sequences were removed to increase the stability of our DNA during synthesis.
The original gene coding for the protein of interest was isolated from a species of Pseudomonas (http://www.ncbi.nlm.nih.gov/nuccore/11890744).
This part encodes a serine protease in Bacillus nematocida target essential intestinal proteins and kill nematode under the control of a constitutive promoter.
We add J23100, a constitutive promoter upstream the coding sequence of bace16, so that it can express Bace16.
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
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.
methylmalonyl-CoA mutase;encodes methylmalonyl-CoA epimerase which catalyzes the reversible reaction of succinyl-CoA and methylmalonyl-CoA
LinA gamma-HCH->1,3,4,6-tetrachloro-1,4-cyclohexadiene
LinA is cofactor-independent dehalogenase from Pseudomonas paucimobilis UT26,and can metabolize gamma-HCH into 1,3,4,6-tetrachloro-1,4-cyclohexadiene to purify water .
No restriction sites contained in the sequence.
LinA is from Pseudomonas paucimobilis UT26
glutamate decarboxylase A （GadA）
The gene GadA (glutamate decarboxylase A) can be cloned from Escherichia coli str. K-12 substr. GadA is a functional gene for producing alkali. It catalyzes the proton-consuming conversion of glutamate to γ-amino butyric acid (GABA) whereas it converts glutamate to GABA by removing the α-carboxylate group of glutamate.
Escherichia coli str. K-12 substr. Sequence found through Genbank http://www.ncbi.nlm.nih.gov/nuccore/NC_000913.3?report=genbank&from=3666180&to=3667580&strand=true
Alkaline phosphatase coding sequence (no rbs, promoter, or terminator present) from Citrobacter. This bacterium's phoA was chosen due simply to the absense of Biobrick restriction sites.
pre-pcr ca1033F/ca1032R on Citrobacter gen (1500 bp) PCR ca1032F/R on above PCR (1500 bp, XbaI/EcoRI) Sub into pSB1A2-Bca1008 (XbaI/MfeI, 2077+935, large) Product is pSB1A2-Bca1032 ---- ca1032F Forward oligo to biobrick phoA of Citrobacter GGTTAtctagaTGTTTTTTATTTTAACTGTATTTATACATGGAG ca1032R Reverse oligo to biobrick phoA of Citrobacter cagtaGAATTCccagaggcggcaTTACTTCAG ca1033F Forward constant oligo to clone phoA ttatcatcgctccgcatcctg
LadA, a long-chain alkane monooxygenase from Geobacillus thermodenitrificans NG80-2, facilitates the conversion of long-chain alkanes (up to at least C36) to the corresponding primary alcohols.
Nucleotide sequence optimized for expression in E.coli
ALDH, an aldehyde deyhydrogenase that facilitates the third step in alkane degradation, from n-alkanals to n-alkanoic acids, which can then be further degraded through β-oxidation
GldA - glycerol dehydrogenase from Escherichia coli with P lacI and RBS
Xuenian chen, Zewei yan and Yichong wang
α-ALS gene express acetolactate synthetase that contribute to produce diacetyl. The synthetize pathway of diacetyl, in E.coli, beginning with the central pathway metabolite pyruvate as the precursor molecule, the first step in the diacetyl biosynthetic pathway involves the condensation of two molecules of pyruvate to yield one molecule of acetolactate, and is catalyzed by acetolactate synthase (ALS).The stability of acetolactate is known to be affected by the presence of oxygen, which causes its spontaneous decarboxylation to produce diacetyl.
LacZ alpha fragment; complements matching N-terminal deletion mutant (lacZ-omega)
to greatly reduce the number of bases, this is only a portion of the LacZ gene
Restriction sites (modified) 76-79 (gcc to gca) 79-81 (gct to gca) 85-87 (gaa to gag) 106-108 (tgc to tgt) 109-111(agg to aga)
medium strong constitutive promoter + luc
This is a firefly luciferase gene under the control of a medium strong constitutive promoter.
medium strong promoter, strong RBS, used this to demonstrate that our luciferase BioBrick is working
* promoter and RBS from partsregistry * luciferase from pGL4.10luc2 (Promega)
This BioBrick BBa_K527000 encode the plant enzyme Cytochrome P450 CYP79A2. Cytochromes p450 are defined as heme-thiolate proteins featuring a particular spectral signature at 450 nm, thus the name. Despite the label as cytochromes, these proteins are not involved in electron transfers, but act as monooxygenases in a wide range of reactions such as epoxidation, N-dealkylation, O-dealkylation, S-oxidation and hydroxylation. CYP79A2 is an N-hydroxylase converting L-phenylalanine into phenylacetaldoxime, the precursor of the cyanogenic glycoside benzylglucosinolate. Oximes as the phenylacetaldoxime is generally toxic to fungi and is frequently used as chemical fungicides. They function by inhibiting mitochondrial oxidases and thereby promote lipid peroxidation and the production of toxic reactive oxygen species.
Mutation at a Pst1 restriction site (nucleotide 343).
Institute of Plant Biology and Biotechnology Faaculty og LIFE Science Copenhagen University Denmark
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.
a gene encoding thermostable alpha-L-arabinofuranosidase from ArfB of Clostridium stercorarium DSM8532, codon optimized for E.coli
whole genome sequencing of Clostridium stercorarium DSM8532
this biobrick is constructed by putting modified enzyme luciferase under promoter T7 and controlled by lac operator. 6 AGG codons and 2 GCG codons are inserted after the ATG start codon of wild type luciferase (BBa_I712019). Modified luciferase keeps the activity of converting luciferin into oxyluciferin, during which bioluminescence will emit. This part is one of the reporter genes to testify the influence of different number of rare codons in regulating protein biosynthesis. This part is used as a measurement to testify the function of LacI -Ptac-tRNAArg (BBa_K567001) or sulA promoter-tRNA(Arg) (BBa_K567002). Cell is cultured in 50ug/ml kanamycin 10ug/ml tetracycline LB liquid medium. When the OD600 of the culture reaches 0.3 IPTG is added to make the final concentration 0.5nM to induce the synthesis of tRNA. Ultrasonication is used to release the luciferase from the cell. Sonics ON 3 seconds, OFF 3 seconds, total ultrasonication time 3minutes. Amount of bioluminescence produced can be detected using luminometer.
PCR is used to insert the 6 AGG codons into the gene after ATG.
T7 promoter is derived from pET28a. Wild type Luciferase from BBa_I712019.
β-lactamase promoter-Luciferase with two AGG-codon insertions. This biobrick is constructed by putting modified enzyme luciferase under constituitive promoter β-lactamase promoter. 2 AGG codons and 2 GCG codons are inserted after the ATG start codon of wild type luciferase (BBa_I712019). Modified luciferase keeps the activity of converting luciferin into oxyluciferin, during which bioluminescence will emit. This part is one of the reporter genes to testify the influence of different number of rare codons in regulating protein biosynthesis. This part is used as a measurement to testify the function of LacI -Ptac-tRNA(Arg)(BBa_K567001) or sulA promoter-tRNA(Arg) (BBa_K567002). Cell is cultured in 50ug/ml kanamycin and 10ug/ml tetracycline LB liquid medium. When the OD600 of the culture reaches 0.3 IPTG is added to make the final concentration 0.5nM to induce the synthesis of tRNA. Ultrasonication is used to release the luciferase from the cell. Sonics ON 3 seconds, OFF 3 seconds, total ultrasonication time 3minutes. Amount of bioluminescence produced can be detected using luminometer.
Point mutation is used to obtain this part from wild type
β-lactamase promoter is derived from pUC18 β-lactamase operon. Wild type Luciferase from BBa_I712019
botin-independent propionyl-CoA carboxylase;a succinate (or propionate) CoA ligase, or a novel (biotin-independent) propionyl-CoA carboxylase
mutation of C to G to avoid illegal PstI site
encodes endo-1,4-beta-xylanase by XynB from Clostridium stercorarium DSM8532, codon optimized for E.coli
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.
This is a modified BBa_I746380 sensitivity tuner. Instead of a GFP reporter, this one has a firefly luciferase (from Promega's pGL4.10luc2 vector) as one reporter gene. The arabinose promoter has been removed from the original part so now it is possible to assemble this part behind any promoter to get an enhanced luciferase signal.
arabinose promoter removed, GFP exchanged with luciferase compared to the original part BBa_I746370
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.
Using different RBSs so that secondary structures and self pairing don't occur.
Composite of genomic sequences coding for proteins involved in e. coli glycogen biosynthesis.
Fusion Protein of S-Layer SgsE and Oxidoreductase
Fusion Protein of S-Layer SgsE and Oxidoreductase;
Naoki Saito,Daiki Haraguchi,Yoshiharu Otaki,Ryuhei Minei
Yichong wang, Ruichen, Xihao liao and Junjie zhang
BBa_K1072002 is the generator fo NADH dehydrogenase which is an enzyme following chemical reaction: NADH + H+ acceptor \rightleftharpoons NAD+ + reduced acceptor
kanamycin resistance backwards (KanB) [cf. BBa_J23012 & BBa_J31003]
Codes the reverse of the Kanamycin Resistance gene. When the region is flipped, Kanamycin resistance will be expressed as long as a promoter and RBS are in front of the region.
HheCW249P 2,3-DCP(2,3-dichloropropanol)->Glycerol
HheC displayed high regioselectivity and moderate to high enantioselectivity that can be applied for the kinetic resolution of chiral spiroepoxides.And HheCW249P is mutant of HheC ,displayed higher activity than the wide type .
1-aminocyclopropane-1-carboxylate (ACC) oxidase
This is the protein coding sequence of 1-aminocyclopropane-1-carboxylate (ACC) oxidase, which catalyses the formation of ethylene from 1-aminocyclopropane-1-carboxylate under the presence of ascorbate and O2. (1-aminocyclopropane-1-carboxylate + ascorbate + O2 = ethylene + cyanide + dehydroascorbate + CO2 + 2 H2O) This enzyme is commonly referred to as ACC oxidase or ethylene forming enzyme (EFE).
Codon optimization for E. coli and removal of biobrick restriction sites.
Solanum lycopersicum (Tomato) (Lycopersicon esculentum) codon optimized for E. coli.
dnMyD88 is a dominant negative form of protein MyD88 (an important protein in signaling cascade through TLR4 receptor), which was our main target for modifying the signaling path. dnMyD88 is linked to Rluc (which acts as a reporter) through six aminoacid long protein linker.
dnMyD88-linker-Rluc was made by using PCR ligation technique. This part is cloned in BioBrick vector pSB1AK3.
nuclear 5-aminolevulinate synthase gene from S. cerevisiae, recoded for mitochondrial expression
The first step in S. cerevisiae's pathway for heme biosynthesis is catalyzed by the product of the HEM1 gene [http://db.yeastgenome.org/cgi-bin/locus.pl?locus=HEM1]. HEM1 is a nuclear gene (1.647 kb,[http://db.yeastgenome.org/cgi-bin/getSeq?seq=YDR232W&flankl=0&flankr=0&map=a3map]) that encodes the enzyme 5-aminolevulinate ("ALA" or "d-ALA") synthase (549 aa,[http://db.yeastgenome.org/cgi-bin/getSeq?seq=YDR232W&flankl=0&flankr=0&map=p3map]). The enzyme is post-translationally imported into the mitochondria where it is responsible for converting glycine and succinyl-CoA to 5-aminolevulinate ("d-ALA") plus CoA and CO2. Deletion of HEM1 is lethal for the cell but viabilty can be restored by providing the cells with the product of the HEM1p catalyzed reaction, namely d-ALA. In an effort to directly express the HEM1 gene from the mitochondrial genome, the HEM1 gene was recoded by DNA 2.0 for mitochondrial codons, an epitope tag (HA = YPYDVPDYA) was added to the C-terminal portion of the protein just before the stop codon, and flanking sequences (100 bp upstm of ATG and 92 dwstm of stop) were added to facilitate integration into mitochondrial genome.
codon optimized for mitochondrial gene expression, HA-epitope C term (not N-term where mt import sequences rest), flank coding seq with homology to COX3 gene of mitochondria to allow for integration and disruption of this gene (should make respiration+ cells become respiration-), and finally added BB prefix and suffix to allow cloning into std Registry vectors
direct synthesis from DNA 2.0, summer 2006
This is a firefly luciferase gene under the control of a weak constitutive promoter.
weak promoter, strong RBS, used this to demonstrate that our luciferase BioBrick is working
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.
This composite part, designed and submitted by the 2016 Broad Run iGEM Team, contains an gene from the bacterium B.cereus that codes for a beta amylase enzyme. This composite part contains the coding sequence, kozak sequence, a mating factor secretion tag, and a terminator. It does not include a promoter. The amylase enzyme can be expressed by cloning this part into a plasmid with a promoter built in (such as the S.cerevisiae yeast vector pAG36).
Codon optimized for S.cerevisiae, Kpn and Spe sites removed.
It is a composite part, coding sequence is sourced from the B.cereus genome.
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.
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.
This part is a composite of RiboJ and I13504 which can be used to express GFP if added a promoter.
In order to make other team easily use a GFP part to test the strength of a promoter, we constructed this part contain RiboJ before the BBa_I13504. Any team can use this part to measure the GFP expression if added a promoter.
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.
This is the pyruvate oxidase from Streptococcus pneumoniae R6. It catalyzes the formation of hydrogen peroxide by oxidizing pyruvate in the reaction: pyruvate + Pi + O2 --> acetyl-P + CO2 + H2O2.
Removal of a PstI site very near the 5' end with PCR primers.
Genomic PCR from a prepared genome of Streptococcus pneumoniae R6 from ATCC (# BAA-255D-5)
α-ALS gene placed under the control of Lux pR promoter. After co-transforming with BBa_K1072000 into E.coli cells,the luxR and AHL complex will activate the Lux pR promotor and transcript α-ALS and GFP genes.
Autoinducer synthetase for acyl-homoserine lactones (AHL): LuxI synthesizes AHL (3OC6HSL). Two molecules of AHL form a complex with two molecules of the LuxR protein. This complex then binds to a palyndromic site on the lux promoter, increasing its transcription rate. LuxI, LuxR and the right lux promoter are all derived from V. fischeri. In absence of AHL, the right lux promotor only gives weak constitutive expression of downstream genes. The LuxI gene is fused to a His-tag facilitating gene expression measurements.
Biobrick BBa_C0061 still contained a Barcode sequence which was deleted. Also, a Ribosome Binding Site was added. For our own use, 5 bp were exchanged to create unique restriction sites and to optimize DNA folding.
Sequence is originating from V. fischeri. Here, the sequence from BBa_C0061 was used and optimized.
Rluc-linker-PEST191 is a part composed of Rluc (which acts as a reporter) linked with PEST191 through six aminoacid long protein linker. PEST191 is a sequence that leads towards degradation of protein linked with this sequence.
Rluc-linker-PEST 191 was made by using PCR ligation technique. This part is cloned in BioBrick vector pSB1AK3.
T7 promoter-metG(truncated). This biobrick is constructed by putting the truncated metG (Met-RS) under the control of T7 promoter and lac operator. We have cloned metG from E.coli and the tRNA recognition domain of metG is truncated. Kana gene with start codon substituted for CGA is used to testify the function of mutated metG. When this biobrick and metY-CGA (BBa_K567016) are co-transformed into the cell, the cells can survive on the LBKana plate.
aspartyl aminoacyl tRNA synthetase without anticodon recognition domain under the control of T7 promoter and lac operator. This biobrick is constructed by deleting the anticodon recognition domain of AspRS from E.coli. This modified AspRS can charge Asp to tRNA(Asp)-TAG (BBa_K567013) and tRNA(Asp)-AGG (BBa_K567012).
Use Dicovery Studio to analyze the enzyme and find the tRNA anticodon recognition domain of AspS.
aspartyl aminoacyl tRNA synthetase derived from AspS in E.coli
LsrK of LuxS/AI-2 signaling pathway in Salmonalla
Quorum sensing is a process of bacterial cell-to-cell communication involving the production and detection of extracellular signaling molecules called autoinducers. And autoinducer-2 (AI-2) has been proposed to serve as a 'universal signal' for interspecies communication. In the LuxS/AI-2 signaling system of Salmonella Typhimurium, AI-2 response involves ATP binding cassette transporter encoded by genes named Lsr (LuxS regulated). And LsrK is the kinase which catalyzes the phosphorylation of autoinducer 2 (AI-2) to phospho-AI-2. In our project, we set this protein-coding part under a nisA promoter and try to integrate them in the genome of Lactobacillus or Lactococcus for the final purpose of constructing an integrated AI-2 response pathway of Salmonella in the engineered bacteria.
The lsrK gene was isolated from the genome of Salmonella enterica subsp. enterica serovar Typhimurium str. LT2 using PCR. We added restriction enzyme sites when designing primers, and the assembly is done through the use of restriction sites (cutting and ligating).
We extracted the genomic DNA of Salmonella enterica subsp. enterica serovar Typhimurium str. LT2, and used PCR to isolate the gene LsrK(Gene ID: 1255599).
7-ketocholesterol is the result of the oxidation that exists in the bloodstream. 7-keto group is being reduced by Oxoacyl Reductase into 7-OH. The identity of this enzyme has not yet veryfied. This nucleotidic sequence was based on Jacques Mathieu studies.He identified gene expression analysis using microarrays and in silico amino acidic predictions.
Codon optimization for a better expression on E.coli.
ATGAAACGCCTGGTCTGTGTTCTGCTGGTCTGCTCGTCTGCTGTTGCTCCGCTGCATCATAGCCTGGTTGATCAC CCGTCTGCCCATCCGGGTCATGGCGGTGGCCCTGTTAGTGCGGGCGTCGCCCTGGTGACCGGTGGCTCCCGTGGC GTTGGTAAAGGCGTCGCAACGGCACTGGGTGCGGCGGGTTGGACCGTGTATGTTACGGGTCGTGGCCCGATGACC GAAGATTGCCCGCTGAGTGCATCCGCTCGTACCGTTACGGGTGCGGGTGGCACCGGTATTGCAGTCGCCTGTGAC CACCGTGAAGATGCAGACGTGCGTGCACTGTTTGCTCGCATCAGTGATGAACAGGCCGGTCGCCTGGACCTGCTG GTTAACAATGTCTGGGCAGCTCCGAAAGGCTTTGGTGGCTTCAGCACCCCGTTCTGGGAACGTCCGCTGGATGAC TGGGATTCTCTGATTGGTGTTGGCCTGCGTGCGCATTATGTGGCCTCAGTTGAAGCAGCACGTCTGATGGTGCCG CGTCGCTCGGGTCTGATTGCGAACATCAGCTCTTTTGGTACCCGCGGCCATCTGCACTCAGTGCTGTACGGCATG TCGAAAGCAGGCCTGGATAAAATGGCTAGCGACATGGCAGCTGAACTGGCCCCGCACGATGTCCGTGCAGTGAGT CTGTGGCCGGGCATTGTGCGCACCGAAATGATCGTTGCGTCCGGTCTGGAAACGATCGCCGGCTTTAGCGTTGCA GATGCTGAATCTCCGGAATTTATTGGTGAAGTCATCTGCGCACTGACCGCCGACCCGACGCTGCCGCAGCGTTCT GGTCATACCTTCGTCACGGCAGAACTGGCTGTGGAATACGGTATTACCGATACGCAAGGTCGTACCCCGCCGAGC CACCGTGAACCGTTTGGTGGCGGTCCGCTGTTCAATATCACG
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
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]
AID (Activation Induced Deaminase)is active in mammal lymphocytes, where it causes the somatic hypermutation ??? an increase of mutation level in antibody coding sequences.
Ref. http://www.ncbi.nlm.nih.gov/pubmed/12097915?ordinalpos=1&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_DefaultReportPanel.Pubmed_RVDocSum
FadA protein coding sequence for fatty acid degradation
For a E.coli to degrade fatty acid, at least four enzymes ??? FadA, FadB, FadD, FadE and transporter???FadL are needed. Long chain fatty acids are firstly being imported by the transmembrane protein FadL. After FAs get into cells, they will be added a CoA by inner membrane-associated FadD (acyl-CoA synthase). β-oxidation is initiated by FadE(acyl-CoA dehydrogenase), which will convert acyl-CoA into enoyl-CoA. The followed cycles of hydration, oxidation, and thiolytic cleavage are carried out by tetrameric complex consisting of two FadA and two FadB monomers. This part is FadA coding sequence, which has 3-ketoacyl-CoA thiolase activity involving in the β-oxidation of living E.coli. The protein takes a part in the planning system to improve whole efficiency of fatty acid degradation. With appropriate promoters we decided, FadA expression can be under regulation of fatty acid concentration.
Before constructing the final system and testing its function, we constructed three biobrick parts with 3-ketoacyl-CoA thiolase activity (FadA, FadI, S-FadA) as candidates.
Stephen Lee, Roshan Kumar, Joe Levine, Ziyan Chu (Polkadorks, IAP 2004)
Coding sequence for AspA enzyme. AspA aminates fumarate to make aspartate. Aspartate can be used as a bacterial chemotaxis signal.
The start codon was changed from GTG to ATG to match BioBricks standards. Five silent mutations were introduced to eliminate EcoRI and PstI restriction enzyme sites. EcoRI sites were eliminated at bases 588 and 657 with C -> T mutations. PstI sites were eliminated at bases 624, 999, and 1125 with G -> A mutations. The mutations were designed to match codons with similar frequencies of usage in E. coli.
This is one of two catalases from E. coli. It has a C-term LAA ssrA tag.
Site directed mutagenesis to remove an EcoRI site.
This is a modified BBa_I746390 sensitivity tuner. Instead of a GFP reporter, this one has a firefly luciferase (from Promega's pGL4.10luc2 vector) as one reporter gene. The arabinose promoter has been removed from the original part so now it is possible to assemble this part behind any promoter to get an enhanced luciferase signal.
luciferase: luciferin 2-monooxygenase from Renilla reniformis (EC 1.13.12.5; SwissProt:: P27652)
Rluc is a protein called Renilla`s luciferase and it emits light when adding the right substrate. That is why it can bi used as a reported to track other proteins or to monitor the activity of a promotor.
Rluc is cloned in BioBrick vector pSB1AK3.
Arabinose regulated RBS-PhoA-His6tag-pNBEst13-Myctag-EstA
Marie Burghard, Jascha Diemer, Philipp Rottmann, Rene Sahm, Arne Wehling
This a a composite of 2 different BioBricks by an standard assembly, forming a scar in between. It contains the following units: AraC-AraO2-AraO1-AraPromo(PBAD)- RBS-PhoA-His6tag-pNBEst13-Myctag-EstA which is similiar to BBa_K808000 & BBa_K808030 Its a regulatory system, being induced by adding 1 to 2% of mw% L-Arabinose. It is made for regulating the cell surface Expression of our PET cleaving enzyme pNB-Est13, which is anchored c-terminal to EstA.
We were not able to add this part as an composite due to missing links and unedited registry pages. Necessary informations are featured in the sequence.
assembled GENEART synthesized product(BBa_K808030)and cloned regulatory unit (BBa_K808000), deriving from an expression system (pBAD-vector)
GldA - glycerol dehydrogenase from Escherichia coli with medium Anderson promoter and medium RBS
Codes for a urease gene, seems to include a promoter.
HyaC is a subunit of hydrogenase 1 (Hyd-1) from Escherichia coli genome. The hya operon, which contains gene for the two HYD1 structual subunits and four additional genes (HyaA-F) was mapped at 22min on the E.coli chromosome. And hyaC is the third gene of hya operon, sharing the first few base pairs with hyaB and the last few base pairs with hyaD. HyaC is a cytochrome that anchors the [NiFe]-hydrogenase to the membrane.
Repression of heat shock gene expression (ROSE) element The repression of heat shock gene expression (ROSE) element is an RNA element found in the 5' UTR of some heat shock protein's mRNAs. The ROSE element is an RNA thermometer that negatively regulates heat shock gene expression. The secondary structure is thought to be altered by temperature, thus it is an RNA thermometer. This structure blocks access to the ribosome binding site at normal temperatures. During heat shock however, the structure changes freeing the ribosome binding site and allowing expression to occur.[2][3] A partial structure of this RNA element has been determined using NMR.[1] ROSE1 and ROSEAT2 are specific examples of ROSE elements.[4] ROSE1 is found in Bradyrhizobium japonicum whereas ROSEAT2 is a closely related element from Agrobacterium tumefaciens. The two RNA elements have similar secondary structures with ROSE1 having an extra hairpin.[4] All ROSE elements contain a characteristic 'bulged G' opposite the Shine-Dalgarno sequence binding site, without this nucleotide the RNA thermometer loses its temperature-sensitivity.[5]
Note that RNA thermometer actually works as an RBS sequence. Therefore, this part does not include an additional ribosome binding site. Also, this part does not contain an terminator sequence, but a simple stop codon. The RFP should be translated continuously.
Expresses glgC mutant gene (coding for the glucose-1-phosphate adenylyltransferase) and glgA gene (coding for glycogen synthase).
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)
Fusion Protein of S-Layer SbpA and NADP+ Oxidoreductase
This is the protein genertors of Rci site-specific recombinase BBa_K361000, which is being linked with a promotor and a ribosome binding site in front of it, a double terminator after it.
This is the optimal system for Rci site-specific recombinase production.
The promotor is come from lacI regulated one: BBa_R0010 The ribosome binding site is come from the defines RBS efficiency equal to one : BBa_B0034 The terminator is come from a doble terminator with bidirectional termination: BBa_B0014
Biobrick BBa_C0061 still contained a Barcode sequence which was deleted. For our own use, 5 bp were exchanged to create unique restriction sites and to optimize DNA folding.
metK (from Streptomyces avermitilis, increasing avermectin production)
T7 RNA polymerase with tyrosine amber codons
quick change mutagenesis on T7 (BBa_K145001)
IaaM encodes IAA tryptophan monooxygenase which converts tryptophan to indoleacetamide
This biobrick is constructed by putting modified enzyme luciferase under promoter T7 and controlled by lac operator. 2 AGG codons and 2 GCG codons are inserted after the ATG start codon of wild type luciferase (BBa_I712019). Modified luciferase keeps the activity of converting luciferin into oxyluciferin, during which bioluminescence will emit. This part is one of the reporter genes to testify the influence of different number of rare codons in regulating protein biosynthesis. This part is used as a measurement to testify the function of LacI -Ptac-tRNA(Arg) (BBa_K567001) or sulA promoter-tRNA(Arg) (BBa_K567002). Cell is cultured in 50ug/ml kanamycin 10ug/ml tetracycline LB liquid medium. When the OD600 of the culture reaches 0.3 IPTG is added to make the final concentration 0.5nM to induce the synthesis of tRNA. Ultrasonication is used to release the luciferase from the cell. Sonics ON 3 seconds, OFF 3 seconds, total ultrasonication time 3minutes. Amount of bioluminescence produced can be detected using luminometer.
PCR is used to insert the 2 AGG codons into the gene after ATG.
Coding region for the 3-hydroxybutyryl-CoA dehydratase (Crt), an enzyme involved in the butyrate biosynthesis pathway of Clostridium acetobutylicum. This substrate of this enzyme is 3-hydroxybutyryl-coA and the product is Crotonyl-CoA.
C.acetobutylicum sequence found through NCBI: http://www.ncbi.nlm.nih.gov/nuccore/NC_003030.1?report=fasta&from=2835666&to=2836451&strand=true
AlkR is a transcriptional regulator belonging to AraC/Xyls family, which could detect a broad range of alkanes and alkenes with carbon chain length from C7 to C36. It is said that AlkR is the only bioreporter that is able to detect alkane with carbon chain length greater than C18.
AlkR with a weaker promoter and terminator.
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
LadA Protein Generator (High expression)
In order to work,Cph8 has to form chromophore with PCB biosynthetic genes, where the formation of PCB requires the gene pcyA and hol to function as accounted below. Hol is a sort of Iron red pigment oxidase which can oxidize the heme group using a ferredoxin cofactor, generating biliverdin IXalpha. And then, PcyA, a kind of ferredoxin oxidase from Synechocystis Sauv, functions to turn biliverdin IXalpha (BV) into PCB. We connected gene pcyA（BBa_I15009）and ho1（BBa_I15008）together along with the constitutive promoter(BBa_J23100) to ensure a continuous work of the photoinduction system in our E.coli.
We used oe-pcr to assemble these parts together seamlessly.
pcyA gene and ho1 gene both come from Synechocystis.
aadA (streptomycin 3'-adenyltransferase)
This part codes for an aminoglycoside resistance protein that endows ''E. coli'' with resistance to the antibiotic streptomycin. This part can be used as a selection marker.
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.
aadA was isolated from a plasmid in ''E. coli''. The original sequence was obtained from GenBank (VERSION NC_002525.1, GI:10955438).
Inducible High Expression His-Tagged Photosystem II CP47 subunit
This part is dddD gene from the genome of Ruegeria pomperoyi. This gene code DddD protein which catalyze breakdown of DMSP and create DMS.
There was PstⅠ recognition site so I made single base substitution in recognition site.
The phoA enzyme without start or stop codons. ---- This part is in BBb Format. It is flanked by BamHI and BglII sites instead of XbaI and SpeI. More information about the BBb Format is available at: [http://openwetware.org/wiki/Template:AndersonLab:BBb_Standard BBb Standard Description Page]
Construction of {<phoA>} Biobrick Part K112233 PCR mea036/mea037 on pBca9145-Bca1126 (1219 bp, EcoRI/BamHI) Sub into pBjh1601AC-Bca1144 (EcoRI/BamHI, 3195,910 L) Product is pBjh1601AC-K112233 ---------------------------------------- mea036 Forward Biobricking of {<phoA>} cgatagaattcatgAGATCTGGAGATTCTG mea037 Reverse Biobricking of {<phoA>} cgttaGGATCCCTTCAGGCCCAGCGCCGCTTTCATG
HyaE is a subunit of hydrogenase 1 (Hyd-1) from Escherichia coli genome. The hya operon, which contains gene for the two HYD1 structual subunits and four additional genes (HyaA-F) was mapped at 22min on the E.coli chromosome. And hyaE is the fifth gene of hya operon, sharing the first few base pairs with hyaD and the last few base pairs with hyaF. HyaE is a hydrogenase maturase. It is important to note that hyaE is not required for the assembly of standard O2-sensitive hydrogenases (Hyd-2 and others) and are apparently only involved in the assembly of O2-tolerant respiratory enzymes.
2-ketobutyrate formate-lyase/pyruvate formate-lyase 4. It catalyzes the reversible conversion of pyruvate and coenzyme-A into formate and acetyl-CoA; the formate can then be used for Hydrogen production. It is activated under anaerobic conditions, we wanted to constitutively express it in order to improve the formate production.
It was amplified by PCR using those primers : CCCGAATTCGCGGCCGCTTCTAGATGAAGGTAGATATT CCCCTGCAGCGGCCGCTACTAGTATCAGAGCGCCTGGGT
It comes from the genome of Escherichia coli K-12 substr. MG1655
kanamycin resistance forward (KanF) [cf. BBa_J23012 & BBa_J31002]
If there is a promoter and an RBS before this region, the cell will express Kanamycin resistance. As it has full biobricks, you can easily add KanR to an existing part without changing the plasmid vector,
lacZ (encoding beta-galactosidase, full-length)
LacZ encodes beta-galactosidase, an intracellular enzyme that cleaves the disaccharide lactose into glucose and galactose. Beta-galactosidase can also cleaves X-gal and ONPG into colorful products.
PCR amplified and standardized from BL21
Erythromycin Esterase Type II (EreB) in RFC[25]
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
β-lactamase promoter-Luciferase with six AGG-codon insertions. This biobrick is constructed by putting modified enzyme luciferase under constituitive promoter β-lactamase promoter. 6 AGG codons and 2 GCG codons are inserted after the ATG start codon of wild type luciferase (BBa_I712019). Modified luciferase keeps the activity of converting luciferin into oxyluciferin, during which bioluminescence will emit. This part is one of the reporter genes to testify the influence of different number of rare codons in regulating protein biosynthesis. This part is used as a measurement to testify the function of LacI -Ptac-tRNA(Arg)(BBa_K567001) or sulA promoter-tRNA(Arg) (BBa_K567002). Cell is cultured in 50ug/ml kanamycin and 10ug/ml tetracycline LB liquid medium. When the OD600 of the culture reaches 0.3 IPTG is added to make the final concentration 0.5nM to induce the synthesis of tRNA. Ultrasonication is used to release the luciferase from the cell. Sonics ON 3 seconds, OFF 3 seconds, total ultrasonication time 3minutes. Amount of bioluminescence produced can be detected using luminometer.
Point mutation is used to obtain this part from BBa_K567005
β-lactamase promoter is derived from pUC18 β-lactamase operon. β-lactamase promoter-Luciferase with four AGG-codon insertions is from BBa_K567005, then point mutated to six AGG-codon insertions
methylmalonyl-CoA decarboxylase;catalyzes the decarboxylation of methylmalonyl-CoA to propionyl-CoA.
E.Coli K12. Sequence found through GeneBank.
This part codes for AdhE protein from ''Clostridium acetobutylicum''. The enzyme is a bi-functional molecule and enables conversion of Acetyl coenzyme A to ethanol.
gDNA of Weizmann Organism: ''Clostridium acetobutylicum''
Coding sequence for the enzyme benzalacetone reductase which reduces 4-hydroxybenzalacetone to raspberry ketone.
Beekwilder, Martinus J., Ole Sibbesen, Joern D. Mikkelsen, Ingrid M. Van Der Meer, Robert D. Hall, and Ingmer Qvist. Synthesis of Benzalacetone/raspberry Ketone by Chalcone Synthase. Danisco, assignee. Patent GB2416770A. 8 Feb. 2006.
CP47 His-tagged with Lac Promoter synthesized by IDT DNA Inc.
yhjH under the control of constitutive promoter J23100
Bordetella pertussis adenylate cyclase subunit T25
One component of a two-hybrid assay. Synthesizes cAMP when it comes in proximity to T18.
Based on the standard Bacterial Adenylate Cyclase Two-Hybrid assay.
PCR amplified from Bordetella pertussis crude cell lysate
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.
This part will prouce a 384-amino acid Rci site-specific recombinase used in shufflon system, which is a site-specific DNA recombination between two specific repeat sites as recognition. After this recombinase is being expressed, it can alter the DNA sequence being inserted randomly with the specific repeat sites originally to become an unknown sequence.
The gene is come from muliple inversion systems in R64 plasmid and there is no further modification. Reports have shown that some modification of the gene will lead to deletion some parts of DNA sequence during recombination process.
It belongs to integrase family of site-specific recombinase, and it is extracted from muliple inversion systems in R64 plasmid, found in genomic sequence code:AP005147.1
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.
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.
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
This is a firefly luciferase gene under the control of a strong constitutive promoter.
strong promoter, strong RBS, used this to demonstrate that our luciferase BioBrick is working
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.
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
the promoter is from iGEM distribution kit and the nadE gene is from the genome of E.coli MG1655
Penicillium alpha amylase w/ no promoter
This composite part, designed and submitted by the 2016 Broad Run iGEM Team, contains an gene from the fungus Penicilium that codes for an alpha amylase enzyme. This composite part contains the coding sequence, kozak sequence, a mating factor secretion tag, and a terminator. It does not include a promoter. The amylase enzyme can be expressed by cloning this part into a plasmid with a promoter built in (such as the S.cerevisiae yeast vector pAG36). The components of this part are: Kozak Sequence - BBa_K165002 This kozak sequence functions as the ribosome binding site for S.cerevisiae. It is involved in translating the sequence from mRNA to amino acids. Mating Factor Secretion Tag - BBa_K792002 This part is the secretion signal peptide from the S.cerevisiae α-mating factor. This signal peptide directs the secretion of the produced protein, allowing the protein to interact with the environment outside the cell. Fungal Alpha Amylase Coding Sequence - BBa_K2185002 This coding sequence codes for the alpha amylase enzyme from the Pencillium fungus. The enzyme can break alpha bonds linking polysaccharides, thus degrading starch into monosaccharides. ADH1 Terminator - BBa_K392003 This is the terminator region from yeast alcohol dehydrogenase (ADH1) gene.
Removed Kpn and spe sites. Codon optimized for S.cerevisiae.
Coding sequence - pencilium fungus (sequence from GenBank)
T7 promoter-metG(mutated). This biobrick is constructed by putting the mutated metG (Met-RS) under the control of T7 promoter and lac operator. We have cloned metG from E.coli and have used error-prone PCR to amplify the metG. Kana gene with start codon substituted for CGA is used to testify the function of mutated metG. When this biobrick and metY-CGA (BBa_K567016) are co-transformed into the cell, the cells can survive on the LB Kana plate.
metG from E.coli. Kana gene derived from pET28a
ecol laccase from E. coli with T7 promoter, RBS and His-tag
E.coli laccase ORF with T7, RBS and HIS tag
linker fused to alpha fragment of TEM beta-lactamase
This is a PPK Coding Sequence which can remove phosphate from water
This is a PPK CDS which is created by Jack Jia from Shenzhen_SFLS and with Eric's great help. The polyphosphatase then degrades phosphates present when there is too much phosphorus in water. We use it as a CDS in our project.
Nothing. Everything goes on well during our research.
N-terminal aminopeptidase (methionine aminopeptidase ??? map gene)
basic part for the chaperone/toxicity device
{{BerkiGEM2007-Biobrick2.0Intro}} N-terminal aminopeptidase (methionine aminopeptidase ??? map gene) Construction of a Biobrick 2.0 basic part in plasmid pBca9145-Bca1144 (Clone # 5) for the N-terminal methionine aminopeptidase (map) open reading frame from Salmonella typhimurium LT2 PCR VU07F.map/VU08R.map on methionine aminopeptidase (817 bp, BglII/XhoI) Digest pBca9145-Bca1144 (Clone # 5) (BglII/XhoI, 2063+910, L) Product is pBca9145-VU3 ------------------------------------- VU07F.map ccaaaAGATCTatggctatctcaatcaagacccc VU08R.map gctagCTCGAGttaGGATCCttattcgtcgtgcgagattatcg
The product of this gene is a phosphodiesterase which is involved in regulating the levels of c-di-GMP, a bacterial signaling molecule. High levels of c-di-GMP promote biofilm synthesis and inhibit flagella by binding to YcgR which then acts as a "brake" on FliG and FliM. Levels of c-di-GMP are elevated and motility is reduced in ''yhjH''-mutants.
The part was amplified from genomic DNA of E. coli str. K-12 substr. DH10B, complete genome (CP000948.1).
NADP+ Oxidoreductase from Escherichia coli
Lux pI+RBS+LuxR+2TM+Lux pR+RBS+LuxI+2TM+Lux pR+RBS+GFP+2TM+Lux pR+RBS+α-ALS+2TM
Junjie zhang, Yichong wang, Zewei yan, Rui chen and Xuenian chen
BBa_K1072007 is a composite divece, including LuxR, LuxI, GFP and α-ALS genes. LuxR is under control of a constitutive promotor, Lux pI. Produce, LuxR, forms a complex with the quorum sensing molecule AHL and this complex subsequently increases the transcriptional rate of the lux pR promoter while also decreas the transcriptional rate of the lux pL promoter. The other genes are under control of this lux pR promoter. The generator, Lux pR+RBS+LuxI+B0015, express AHL which will combine with LuxR. GFP also is under control of Lux pR, presenting as reporter. α-ALS，as our desire produce, will transcrip activated by LuxR and AHL complex. If co-transforming with B0015 , this system will transcript α-ALS periodically. Finally, this part just is the combination of BBa_K1072000 and BBa_K1072006 .
Some part get from registry and α-ALS gene originate from Lactococcus lactis, respectively.
IaaH encodes Indoleacetimide hydrolase which catalyses Indolacetimide to IAA
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.
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.
Zewei yan, Yichong wang and Junjie zhang
BBa_K1072004 is the conding region of α-acetolactate synthetase(α-ALS).
HyaB is the large subunit of hydrogenase 1 (Hyd-1) from Escherichia coli genome. The hya operon, which contains gene for the two HYD1 structual subunits and four additional genes (HyaA-F) was mapped at 22min on the E.coli chromosome. And hyaB is the second gene of hya operon, sharing the first few base pairs with hyaA and the last few base pairs with hyaC. This large subunit of this [Ni-Fe] hydrogenase contains the active site. The large subunit exhibit sequence homologies to subunits of NADH: ubiquinone oxidoreductase (Complex I).
tthl laccase from Thermus thermophilus with T7 promoter, RBS and His-tag
tthl (Laccase from Thermus thermophilus) with T7, RBS and HIS tag
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.
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.
Modified rhlABRI operon genes from Pseudomonas aeruginosa SQ6
PCR sil03F and sil03R on barnase region (348 bp, BglII/XhoI) Sub into pBca9145-Bca1089 (BglII/XhoI, 2063+696, L) Product is pBca9145-I716211 sil03F Forward BglII biobrick of barnase ccaaaAGATCTgcacaggttatcaacacgtttg sil03R Reverse XholI/BamHI biobrick of barnase gctagCTCGAGttaGGATCCttatctgatttttgtaaagg
Catalyzes the following reaction: succinyl-CoA + 3-oxoadipate <<-->> succinate + 3-oxoadipyl-CoA This enzyme consists of a alpha and beta subunit. It is involved in benzoate degradation via hydroxylation.
SOURCE Pseudomonas putida KT2440 ORGANISM Pseudomonas putida KT2440 Bacteria; Proteobacteria; Gammaproteobacteria; Pseudomonadales; Pseudomonadaceae; Pseudomonas. REFERENCE 1 (bases 1 to 696) AUTHORS Nelson,K., Paulsen,I., Weinel,C., Dodson,R., Hilbert,H., Fouts,D., Gill,S., Pop,M., Martins Dos Santos,V., Holmes,M., Brinkac,L., Beanan,M., DeBoy,R., Daugherty,S., Kolonay,J., Madupu,R., Nelson,W., White,O., Peterson,J., Khouri,H., Hance,I., Lee,P., Holtzapple,E., Scanlan,D., Tran,K., Moazzez,A., Utterback,T., Rizzo,M., Lee,K., Kosack,D., Moestl,D., Wedler,H., Lauber,J., Hoheisel,J., Straetz,M., Heim,S., Kiewitz,C., Eisen,J., Timmis,K., Duesterhoft,A., Tummler,B. and Fraser,C. TITLE Complete genome sequence and comparative analysis of the metabolically versatile Pseudomonas putida KT2440 JOURNAL Environ. Microbiol. 4 (12), 799-808 (2002)
β-lactamase promoter-Luciferase with four AGG-codon insertions. This biobrick is constructed by putting modified enzyme luciferase under constituitive promoter β-lactamase promoter. 4 AGG codons and 2 GCG codons are inserted after the ATG start codon of wild type luciferase (BBa_I712019). Modified luciferase keeps the activity of converting luciferin into oxyluciferin, during which bioluminescence will emit. This part is one of the reporter genes to testify the influence of different number of rare codons in regulating protein biosynthesis. This part is used as a measurement to testify the function of LacI -Ptac-tRNA(Arg)(BBa_K567001) or sulA promoter-tRNA(Arg) (BBa_K567002). Cell is cultured in 50ug/ml kanamycin and 10ug/ml tetracycline LB liquid medium. When the OD600 of the culture reaches 0.3 IPTG is added to make the final concentration 0.5nM to induce the synthesis of tRNA. Ultrasonication is used to release the luciferase from the cell. Sonics ON 3 seconds, OFF 3 seconds, total ultrasonication time 3minutes. Amount of bioluminescence produced can be detected using luminometer.
β-lactamase promoter is derived from pUC18 β-lactamase operon. Luciferase with two AGG-codon insertions from BBa_K567004.
encodes alpha-L-arabinofuranosidase ArfB from Clostridium stercorarium DSM8532, codon optimized for E.coli
from whole genome sequenceing of Clostridium stercorarium DSM8532, codon optimized for E.coli
Human guanylate cyclase1,soluble, alpha 3 unit
Soluble guanylate cyclases(sGC) are heterodimeric proteins that catalyze the conversion of GTP to 3',5'-cyclic GMP and pyrophosphate. It is important for smooth muscle relaxation in the cardiovascular system. sGC contains an HNOX domain, which serves as a receptor for ligands such as nitric oxide, oxygen and nitrovasodilator drugs.There are several different isoforms of sGC subunit ,but the cardiovascular system abundant with alpha 3 unit and beta 3 unit.
This subunit coding part with a GFP reporter was then transfected into HEK293 cells and in vivo green fluorescence signal was observed under fluorescence microscope. After that we used lentiviral vector to transfected alpha 3 unit and beta 3 unit to HEK293 cells together. Since soluble guanylate cyclases (sGC) are heterodimeric proteins that catalyze the conversion of GTP to 3',5'-cyclic GMP(cGMP) and pyrophosphate. The level of cGMP will be up regulated.The positive control was HEK293 cells that treat with Sodium Nitroprusside ,a NO donator that activate sGC and up regulate the level of cGMP. A negative control was made by transfecting an empty vector that does not contain sGC subunit. We used Elisa to detect cGMP level.
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
LinB 1,3,4,6-tetrachloro-1,4-cyclohexadiene->2,5-dichloro-2,5-cyclohexadiene-1,4-diol
LinB is cofactor-independent dehalogenase from Pseudomonas paucimobilis UT26,and can do furthur degration of gamma-HCH after LinA's work to purify water .
LinB is from Pseudomonas paucimobilis UT26
Coding region for PyrF gene. PyrF is used in the synthesis of uracil in the same pathway that Ura3 is used in yeast. Can also be used as a conterselectable marker in the presence of 5-Fluoroorotic Acid (5-FOA) and the absence of uracil.
Four silent mutations were introduced to eliminate EcoRI and PstI restriction enzyme sites. The EcoRI site was eliminated at base 39 with a G -> A mutation. PstI sites were eliminated at 240 and and 441 with T -> C mutations, and at 261 with a T -> G mutation. Mutation choices were selected to match codons with similar codon usage.
chloramphenicol acetyltransferase (backwards, CmB) [cf. BBa_J31005]
Codes the reverse of the CmR gene. If the region has a promoter and an RBS in front and is flipped, the cell will express Chloramphenicol resistance.
tthl laccase from T. thermophilus with constitutive promoter J23100, RBS and His-tag
tthl laccase (T. thermophilus) with constitutive promoter and HIS tag
gluc1phos adenylyltrans (with 5'UTR & term)
The sequence provided here is from the CL1136 strain, with an Arg67Cys mutation in the glgC gene.
One of two requisite genes required for the biosynthesis of phycocyanobilin from heme.
HyaF is a subunit of hydrogenase 1 (Hyd-1) from Escherichia coli genome. The hya operon, which contains gene for the two HYD1 structual subunits and four additional genes (HyaA-F) was mapped at 22min on the E.coli chromosome. And hyaE is the sixth gene of hya operon, sharing the first few base pairs with hyaD and the last few base pairs with hyaF. HyaF is a hydrogenase maturase. Similar to hyaE, hyaF is not required for the assembly of standard O2-sensitive hydrogenases (Hyd-2 and others) and are apparently only involved in the assembly of O2-tolerant respiratory enzymes. HyaF interacts with HyaE to form a complex together with the small subunit during assembly.
This part codes for the chloramphenicol acetyltransferase (cat) gene used in Bacillus subtilis for resistance against the antibiotic chloramphenicol. We constructed it in the freiburg standard (Assembly 25) for in-frame fusion-proteins. There is a Shine-Dalgarno Sequence (RBS) optimized for Bacillus subtilis included between the XbaI and NgoIV restriction site. You can use it both N-and C terminal fusions.
We constructed the part in the freiburg standard (Assembly 25). We included a Bacillus optimized RBS between the XbaI and NgoIV restriction site.
Plasmid: Bacillus subtilis reporter vector pAC6
Yichong wang, Zhiwei li, Xihao liao and Junjie zhang
BBa_K1072001 is the coding region of NADH dehydrogenase which is an enzyme following chemical reaction: NADH + H+ + acceptor \rightleftharpoons NAD+ + reduced acceptor
Lux pL controlled LuxR+Lux pR autoinduceing luxI(lva tag)+Lux pR controlled Ndh
BBa_K1072003 is a composite device, including LuxI，LuxR and Ndh genes. LuxR， controlled by Lux pI, generate LuxR protein, which will combined with AHL and activate Lux pR promotor. that complex increases the transcriptional rate of the lux pR promotor while also decrease the transceiptional rate of the Lux pL promoter. LuxI is the generator of AHL controlled by Lux pR promoter. Ndh was activated by AHL and LuxR complex and yields H2O2;
PompR is a OmpR-controlled promoter which can be positively regulated by phosphorylated OmpR. This promoter is taken from the upstream region of ompC. Phosphorylated OmpR binds to the three operator sites and activates transcription. We insert PompR(BBa_R0082) sequences into the upstream region of our target gene gp35 through restriction-ligation method, which together is connected to vector PSB1C3 afterwards, and therefore we are able to regulate the expression of in through the control of light. Bxb1 gp35 is a serine integrase in Mycobacterium phage Bxb1. This intergrase can exclusively catalyze site-specific recombination. The dismiss of light singal can drive the expression of integrase, which alone can set a DNA sequence(there it means the promoter) flanked by oppositional attB and attP sites, thereby produce an inverted sequence flanked by attL and attR sites. As a consequence, the promotor moves on to express the toxic protein instead of the attractant. We insert the gp35 gene into the downstream region of the PompR promotor by means of 3A synthesis so that the expression of integrase can be regulated by light. In addition, we add a 6N promotor between PompR and gp35 to decrease basal integrase expression above flipping threshold level which could lower the efficiency of int translation. Meanwhile, we insert the recombined sites of attB and attP into the flanking sequences of our constitutive promotor (BBa_J23110 ) and so far we accomplish our reverse promotor system.
we add a 6N promotor between PompR and gp35 to decrease basal integrase expression above flipping threshold level which could lower the efficiency of int translation.
This biobrick is constructed by putting modified enzyme luciferase under promoter T7 and controlled by lac operator. 8 AGG codons and 2 GCG codons are inserted after the ATG start codon of wild type luciferase (BBa_I712019). Modified luciferase keeps the activity of converting luciferin into oxyluciferin, during which bioluminescence will emit. This part is one of the reporter genes to testify the influence of different number of rare codons in regulating protein biosynthesis. This part is used as a measurement to testify the function of LacI -Ptac-tRNAArg (BBa_K567001) or sulA promoter-tRNA(Arg) (BBa_K567002). Cell is cultured in 50ug/ml kanamycin 10ug/ml tetracycline LB liquid medium. When the OD600 of the culture reaches 0.3 IPTG is added to make the final concentration 0.5nM to induce the synthesis of tRNA. Ultrasonication is used to release the luciferase from the cell. Sonics ON 3 seconds, OFF 3 seconds, total ultrasonication time 3minutes. Amount of bioluminescence produced can be detected using luminometer.
PCR is used to insert the 8 AGG codons into the gene after ATG.
HyaA is the small subunit of hydrogenase 1 (Hyd-1, from MBH family) from Escherichia coli genome. The hya operon, which contains gene for the two HYD1 structural subunits and four additional genes (HyaA-F) was mapped at 22min on the E.coli chromosome. And hyaA is the first gene of hya operon, sharing the last few base pairs with hyaB. Under carbon starvation upon entering stationary phase, expression of the hya operon, which encodes Hyd-1, is regulated by sigma-38. The small subunit exhibits sequence homologies to subunits of NADH: ubiquinone oxidoreductase (Complex I). This small subunit of this [Ni-Fe] hydrogenase contains 4 Fe-S clusters ([Fe3S4], [Fe4S4]d, [Fe4S3]) that play a role in ET to and from the large subunit for H2 reduction/oxidation. The supernumerary cysteine in the proximal cluster of the small subunit also plays a role in O2 tolerance.
yhjH under the control of constitutive promoter J23114
* ''yhjH'' was amplified from genomic DNA of ''E. coli'' str. K-12 substr. DH10B, complete genome (CP000948.1). * J23112 information was taken from the partsregistry and physical DNA from the 2012 distribution kit.
Our proposed gene sequence is an actuator that produces ADH1B following an RNA polymerase per second (PoPS) signal from a sensor. The sensor will be followed by a bi-cistronic ribosome binding sequence provided by BIOFAB (BD2). This type of ribosome binding sequence contains a ribosome binding site followed by the DNA sequence TAATG, which terminates translation of the first part with stop codon TAA. A second ribosome can bind in the sequence and start translation of the gene with start codon ATG. The bi-cistronic ribosome binding sequence prevents the protein???s ribosome binding site from forming a hairpin loop with another part of the sequence, which would reduce the amount of translation. Specifically, our group chose to use BD2 because it offers consistently high to medium expression of the gene of interest. After BD2, we have inserted the gene sequence of ADH1B, which we constructed using the protein sequence from protein database UniProt that was cross referenced with NCBI. Since we are inserting a eukaryotic gene into a prokaryote, we optimized the DNA sequence for E. Coli using a codon optimizing chart provided by Sharp et al. (1988), selecting for highly transcribed genes as our objective is to produce large amounts of the enzyme. Our ADH1B gene was followed by a histidine tag consisting of 6 histidines that can be used to purify the enzyme with nickel affinity chromatography. Then we put a stop codon TAG that terminates translation. Lastly, we placed a transcription terminator, part apFAB391 provided by Biofab, that terminates transcription 99% of the time because our objective is to transcribe ADH1B so we would want to prevent transcribing other proteins on the same plasmid to conserve energy.
We maximized expression by using a highly expressing bicistronic RBS. We needed a way to purify the enzyme post production. The histidine tag allows us to perform nickel affinity chromatography to isolate purified protein.
Bicistonic RBS - BioFab: apFAB563 ADH1B - partsregistry: BBa_M36666 Histidine Tag - 6 histidines Stop Codon - generic TAG Terminator - BIOFAB: apFAB391
Aad9 is the spectinomycin resistance gene from ''Enterococcus faecalis''#1. Expression in a host confers resistance to spectinomycin at a concentration of 100μg/μl and has been used in a variety of vectors for both ''B. subtilis'' and ''E. coli'' including pDP870#2, pCOMT-Kan#3 and pIEF16s#4 ====References==== #1 pmid=1659306 #2 pmid=16997985 #3 pmid=15060042 #4 pmid=16714443
The sequence of ''B. subtilis'' integration vector pDR111 was searched for the spectinomycin resistance gene and the Biobrick standard applied to the gene sequence upon PCR cloning
Aad9 was PCR cloned from the ''B. subtilis'' integration vector pDR111 using Pfu DNA polymerase
GldA - glycerol dehydrogenase from Escherichia coli with P T7 and RBS
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
Latex Clearing Protein (Lcp) - rubber degradation
Latex clearing protein (Lcp) from actinomycete Streptomyces sp. strain K30 (GenBank accession code: AAR25849.1) is a 42kDa enzyme which catalyzes rubber cleavage at the cis double bonds to multiple products ranging from C20 tetra-isoprenoid to at least C35 hepta-isoprenoid with measured specific activity of 1.3 mol/min per mg.
Because Lcp is secreted by Streptomyces sp, it contains a 5???-signal peptide that was identified using signal peptide predictor server (4) (http://www.compgen.org/tools/PRED-TAT). We removed this region from Lcp to generate this part flanked by SacI and NdeI restrictions sites.
Lcp from actinomycete Streptomyces sp. strain K30
autoinducer inactivation enzyme aiiA (no LVA)
Thermostable lipase (TliA) of Pseudomonas fluorescens SIK W1
Jung Hoon Ahn from Korea Advanced Institute of Science and Technology
TliA has four glycinerich repeats (GGXGXD) in its C-terminus, which appear in many ABC transporter-secreted proteins.Export of fusion proteins with the whole TliA through the ABC transporter was evident on the basis of lipase enzymatic activity. Upon supplementation of E. coli with ABC transporter, EGF-TliA was excreted into the culture supernatant.Whole TliA were attached to C-termini of model proteins and enabled the export of the model proteins such as GFP and EGF which has 3 disulfide bonds in E. coli supplemented with ABC transporter. Activity domain (residues 1???268) and secretion/chaperon domain (residues 279???476). In our experiment, we observed that TliA fused proteins were excreted to supernatant culture succesfully by detecting lipase activity with tributyrin and spectrophotometric detection with the substrate p-nitrophenyl phosphate at 420 nm. At the experiments, Tlia fused proteins were excreted ten-fold more with ABC transporter PrtDEF of Erwinia chrysanthemi.
The sequence was optimized for E.coli with Biobrick restriction sites
The thermostable lipase (TliA) of P. fluorescens SIK W1 is comprised of 476 amino acids and has the characteristic C-terminal signal sequence recognized by the ABC transporter.
bpul (laccase from Bacillus pumilus) with T7 promoter, RBS and HIS tag
bpul (Laccase from Bacillus pumilus) with T7, RBS and HIS tag
Modified rhlAB coding sequence from Pseudomonas aeruginosa SQ6
yddV is a Diguanylate Cyclase-Genomic. The product of gene yddV has diguanylate cyclase (DGC) activity. DGC uses 2 GTP to form a Bis-(3???-5???)-cyclic dimeric guanosine monophosphate (c-di-GMP). C-di-GMP is a global second messenger in bacteria. Biofilm formation of E.coli is manipulable by varying c-di-GMP concentrations. When the c-di-GMP level stays low, there is little biofilm and the bacteria is dispersive. High concentrations of c-di-GMP promote bacteria to form more cellulose and fimbriae, which enhances the biofilm formation and decrease the motility of bacteria.
Butyrate synthesis pathway regulated by constitutive promoter p(Bla)
Constitutive promoter p(Bla). Crotonyl-coA reductase ccr coding region. 3-hydroxybutyryl-CoA dehydrogenase hbd coding region. 3-hydroxybutyryl-CoA dehydratase crt coding region. Acyl-CoA thioesterase 2 tesB coding region. acetyl-CoA acetyltransferase atoB coding region. Every genes are framed by strong RBS based on Ron Weiss thesis. This operon ends with an artificial terminator according to BBa_B1006. Due to a lack of time we decided to have this construction synthesised instead of using the assembly protocol. Unfotunatly NMR analysis did not yield a significant over production of butyrate in E. coli.
p(Bla) is followed by NheI restriction site and ccr is followed by XhoI restriction site
tesB, ccr, crt, and hbd sequence are from Clostridium acetobutylicum. atoB sequence is from E.coli. The strong RBS and Terminator were used according to respectively BBa_B0030 and BBa_B1006
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
this biobrick is constructed by putting modified enzyme luciferase under promoter T7 and controlled by lac operator. 4 AGG codons and 2 GCG codons are inserted after the ATG start codon of wild type luciferase (BBa_I712019). Modified luciferase keeps the activity of converting luciferin into oxyluciferin, during which bioluminescence will emit. This part is one of the reporter genes to testify the influence of different number of rare codons in regulating protein biosynthesis. This part is used as a measurement to testify the function of LacI -Ptac-tRNA(Arg) (BBa_K567001) or sulA promoter-tRNA(Arg) (BBa_K567002). Cell is cultured in 50ug/ml kanamycin 10ug/ml tetracycline LB liquid medium. When the OD600 of the culture reaches 0.3 IPTG is added to make the final concentration 0.5nM to induce the synthesis of tRNA. Ultrasonication is used to release the luciferase from the cell. Sonics ON 3 seconds, OFF 3 seconds, total ultrasonication time 3minutes. Amount of bioluminescence produced can be detected using luminometer.
PCR is used to insert the 4 AGG codons into the gene after ATG.
Celia Chenebault, Camille Soucies, Benjamin Piot
The Pr promoter is a constitutive promoter driving the transcription of the XylR gene, coding for the XylR protein. The XylR protein, a transcriptional regulatory protein for the Pu promoter, is activated by aromatic hydrocarbons that carry a methyl group (like toluene and xylene). In our biosensor the Pu promoter allows the transcription of the bioluminescent reporter gene GLuc, coding for the Gaussia luciferase. When this enzyme reacts with its coelenterazine substrate it emits luminescence.
Site directed mutagenesis to overcome the mutation in the start codon of XylR
IDT synthesis in 3 parts that were then assembled
Luciferase under the control of β-lactamase promoter with TAG insertion. A TAG codon is inserted after the initial codon of the gene. This part is used to testify the function of corporation of PT7-TDRS (BBa_K567011) and tRNA(Asp)-TAG (BBa_K567013).
Point mutation is used to obtain this part from the wild type.
β-lactamase promoter is derived from pUC18 β-lactamase operon. Wild type luciferase is derived from BBa_I712019.
Expresses the glgC, glgA, and glgS proteins/enzymes usually not expressed in e. coli.
The start codon of Kana gene is mutated from ATG to CGA.
In strains with lacZ-omega (lacZ N-terminal deletion mutant) like DH5alpha, DH10B and Top10, lacZ-alpha restores the beta-galactosidase activity.
PCR amplified from BBa_J33202 without the RBS.
pentaerythritol tetranitrate reductase with native rbs
This is the coding sequence for the enzyme pentaerythritol tetranitrate reductase (PETN reductase) of ''Enterobacter cloacae'' PB2. This enzyme reductively denitrates nitrate esters such as PETN (pentaerythritol tetranitrate, penthrite) GTN (glycerol trinitrate, nitroglycerine) and EGDN (ethylene glycol dinitrate) yielding the alcohol and nitrite ion. It can also reductively attack nitroaromatic compounds such as TNT (2,4,6-trinitrotoluene) forming initially hydride-Meisenheimer complexes which break down via an unknown route to yield nitrite and non-aromatic products. Reference: Binks, P.R., French, C.E., Nicklin, S., & Bruce, N.C. (1996). Degradation of pentaerythritol tetranitrate by ''Enterobacter cloacae'' PB2. Applied and Environmental Microbiology 62(4), 1214-1219. Reference 2: French,C.E., Nicklin,S. and Bruce,N.C. 1996. Sequence and properties of pentaerythritol tetranitrate reductase from ''Enterobacter cloacae'' PB2. J. Bacteriol. 178 (22), 6623-6627 (1996).
No special considerations, but due to an error, the primers were actually designed based on the homologous gene of another strain of ''Enterobacter cloacae'', which has a very similar coding sequence but different sequence in the upstream region. The sequence shown here is the correct sequence determined by sequencing the construct.
This part was amplified from plasmid pONR1 which bears the PETN reductase gene originally cloned from ''Enterobacter cloacae'' PB2.
Medium-chain alkane 1-monooxygenase (Gordonia sp. TF6)
Alkane 1-monooxygenase from Gordonia sp. TF6
This composite has an associated RBS (BBa_B0034) to BBa_K1313001.
β-lactamase promoter-Luciferase with 8 AGG-codon insertions. This biobrick is constructed by putting modified enzyme luciferase under constituitive promoter β-lactamase promoter. 8 AGG codons and 2 GCG codons are inserted after the ATG start codon of wild type luciferase (BBa_I712019). Modified luciferase keeps the activity of converting luciferin into oxyluciferin, during which bioluminescence will emit. This part is one of the reporter genes to testify the influence of different number of rare codons in regulating protein biosynthesis. This part is used as a measurement to testify the function of LacI -Ptac-tRNA(Arg)(BBa_K567001) or sulA promoter-tRNA(Arg) (BBa_K567002). Cell is cultured in 50ug/ml kanamycin and 10ug/ml tetracycline LB liquid medium. When the OD600 of the culture reaches 0.3 IPTG is added to make the final concentration 0.5nM to induce the synthesis of tRNA. Ultrasonication is used to release the luciferase from the cell. Sonics ON 3 seconds, OFF 3 seconds, total ultrasonication time 3minutes. Amount of bioluminescence produced can be detected using luminometer.
β-lactamase promoter-Luciferase with 6 AGG-codon insertions is derived from BBa_K567006 then point mutation is performed to obtain this part.
β-lactamase promoter is derived from pUC18 β-lactamase operon. β-lactamase promoter-Luciferase with 6 AGG-codon insertions is derived from BBa_K567006 then point mutation is performed to obtain this part.
this gene encodes endo-1,4-beta-xylanase of XynB from Clostridium stercorarium DSM8532, codon optimized for E.coli
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.
chloramphenicol acetyltransferase (forwards, CmF) [cf. BBa_J31004]
When a promoter and an RBS are in front of the gene, the cell will express Chloramphenicol resistance. Because it contains full biobrick ends, this part can be used to easily add chloramphenicol resistance to any part without changing plasmid vectors.
Truncated lysostaphin coding sequence. Lysostaphin has has a specific lytic action against S.aureus.
Lysostaphin is a zinc metalloenzyme that has a specific lytic action against S.aureus. Lysostaphin has activities of three enzymes namely, endo-β-N-acetyl glucosamidase, N-acteyl-muramyl-L-alanine amidase, glycylglycine endopeptidase. Glycylglycine endopeptidase lyses staphylococcal cells by hydrolyzing glycylglycine bonds in the poly-glycine bridges which form cross links between glycopeptide chains in cell wall peptidoglycan of S.aureus cells. The wild-type lysostaphin gene encodes a preproenzyme, and the conversion of prolysostaphin to mature lysostaphin occurs extracellularly and involves the removal of the hydrophilic tandem repeat portion of the proenzyme. In order to directly produce mature lysostaphin, we truncate the preprolysostaphin and prolysostaphin sequence.
In order to directly produce mature lysostaphin, we truncate the preprolysostaphin and prolysostaphin sequence.
Coding sequence for trunctated Lysostaphin regulated by T7-promoter
Coding sequence for truncated Lysostaphin regulated by T7-promoter. This enzyme has the ability to cleave cross-linking pentaglycine bridges in the cell wall peptidoglycan found in certain Staphylococci.
The subunits were aligned by 3A Assembly.
The sequence encoding for lysostaphin is synthesised de novo according to iGEM Harbin 2012.
Firefly luciferase - luciferase from Photinus pyralis
Firefly luciferase is luciferase from Photinus pyralis. By oxidation of substrate luciferin light is produced. Luciferase thus acts as a reporter protein when connected to other proteins or promoters.
Part is cloned into pSBAK3 vector with terminator.
This firefly luciferase comes from Promega PGL3 vector.
α-amylase coding parts from Aspergillus oryzae.
EMBL-Bank:X12725.1:Aspergillus oryzae amy1 gene for alpha-amylase (EC 3.2.1.1)
bhal laccase from Bacillus halodurans with T7 promoter, RBS and His-tag
bhal laccase from Bacillus halodurans with T7 and HIS
HyaD is a subunit of hydrogenase 1 (Hyd-1) from Escherichia coli genome. The hya operon, which contains gene for the two HYD1 structual subunits and four additional genes (HyaA-F) was mapped at 22min on the E.coli chromosome. And hyaD is the fourth gene of hya operon, sharing the first few base pairs with hyaC and the last few base pairs with hyaE. HyaD is the specific protease required for large subunit maturation-terminal processing. HyaD is a hydrogenase maturase.
In order to have a better expression in E. coli, we optimised the coding sequence of rMpL, and add an L- arabinose induced promoter pBAD.
bifunctional Cyclase from Gibberella fujikuroi
Genomic sequence of Gibberella fujikuroi.