folB is part of the L.lactis folate gene cluster.
We were unsure of whether or not the L.lactis RBS would work with E. coli and so we are going to try cloning the entire gene cluster with the RBSs as well as cloning out each gene separately and adding E.coli RBSs.
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 Biobrick is created by putting enzyme cassette CrtEBI (with individual rbs) of Part BBa_K274100 under constitutive promoter R0011. Enzyme cassette CrtEBI (with individual rbs) of Part BBa_K274110 converts colourless farnesyl pyrophosphate to red lycopene (via intermediates geranylgeranyl pyroiphosphate and phytoene). Amount of lycopene produced can be measured by photospectrometer with absorbance at 475nm (lycopene extraction using acetone).
The colourless precursor, farnesyl pyrophosphate, is naturally produced in E.coli using Non-mevalonate Pathway (from pyruvate and glyceraldehyde-3-phosphate). Our experiences showed that this Biobrick works better in E.coli strains with up-regulated level of precursors. E.coli strain MG1655 demonostrates higher level of pigment (lycopene) production than strain TOP10.
Gene coding sequences come from Pantoea ananatis (formerly Erwinia uredovora) (Accession number D90087), a type of Gram negative Enterobacteriaceae. Please refer to Part BBa_K274100 for more information on individual components.
Diguanylate cyclases produce cyclic diGMP (c-di-GMP), a secondary messenger. Diguanylate Cyclase (DgcZ), also called YdeH, is present in Escherichia coli and the stimulus that controls it is Zinc.
The sequence did not have any restriction sites to remove so it was immediately PCRed out of the genome and had the registry prefix and suffix added to it. It was then ligated into the pSB1C3 vector. YdeH however, is toxic to E. coli cells.
The source of this part is Escherichia coli MC40100 genomic DNA which was provided by the Tracy Palmer lab in the Molecular Microbiology department of the University of Dundee, UK.
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
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]
PabB from L.lactis is a fusion gene of both the usual pabB and pabC in the pABA synthesis pathway from chorismate.
The start codon of pabB in L.lactis is TTG, which cannot be used in E.coli. So we designed the primer such that the first T was omitted and the A from the xba1 primer make an ATG start codon.
rbs-ThpB-rbs-ThpC-rbs-ThpD-TT will help produce hydroxyectoine to withstand freeze dry.
Digest I716340 (BglII/XhoI, 2057bp, 1058bp, S) Sub into I716339 (BamHI/XhoI, 3759bp, 9bp, L) Product is I716341
This Biobrick is created by putting enzyme cassette CrtEBIY (with individual rbs) of Part BBa_K274200 under constitutive promoter R0011. Enzyme cassette CrtEBIY (with individual rbs) of Part BBa_K274200 converts colourless farnesyl pyrophosphate to orange beta-carotene (via intermediates geranylgeranyl pyroiphosphate, phytoene and lycopene). Amount of beta-carotene produced can be measured by photospectrometer with absorbance at 455nm (beta-carotene extraction using acetone). Sequence and Features
Gene coding sequences come from Pantoea ananatis (formerly Erwinia uredovora) (Accession number D90087), a type of Gram negative Enterobacteriaceae. Please refer to Part BBa_K274200 for more information on individual components.
Test plataform for rhamnosyltransferase BioBrick (Rh1AB_BB) expression in E. coli
Ammonium monooxygenase from Nitrosomonas europaea
LacI regulated ethylene generator that uses 3 enzymes, blah
This composite part is made of a standard ribosomal binding site and a part that codes for a polyhydroxybutyrate (PHB) synthase, which catalyzes the polymerization of hydroxybutyryl-CoA.
PCR using primers with biobrick prefix and surfix, and then cloned into pSB1AC3.
rbs-ThpB-rbs-ThpC will help in the production of hydroxyectoine to withstand freeze dry.
Digest I716337 (BglII/XhoI, 2057bp, 428bp, S) Sub into I716336 (BamHI/XhoI, 3340bp, 9bp, L) Product is I716339
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
folKE (GTP Cyclohydrolase I + pyrophosphokinase)
folKE is a bifunctional gene which encodes two necessary enzymes for folate biosynthesis (2-Amino-4-hydroxy-6-hydroxymethyldihydropteridine pyrophosphokinase / GTP cyclohydrolase I).
IAA biosynthetic genes under control of the Pveg2 promoter
The construct of IaaM and IaaH genes in PVEG plasmid
Galactose oxidase is a fungal enzyme that uses molecular oxygen to oxidizes galactose and several other primary alcohols to the corresponding aldehyde, while also producing hydrogen peroxide. This particular galaxtose oxidase is not wild type. It has been optimized through directed evolution by the Arnold lab at CalTech to be functionally expressed in vivo in E. coli and to be thermostable. (Sun L, Petrounia IP, Yagasaki M, Bandara G, and Arnold FH. Expression and stabilization of galactose oxidase in Escherichia coli by directed evolution. Protein Eng 2001 Sep; 14(9) 699-704. pmid:11707617). This parts has undergone two rounds of site directed mutagenesis to remove one EcoRI and one PstI site. In our hands, it is functional towards galactose in vivo.
Two rounds of site directed mutagenesis had to be performed to eliminate an EcoRI and PstI site.
Plasmid from the Arnold lab at CalTech, which is the result of their directed evolution to make galactose functionally expressed in E. coli and to improve its thermostability.
Rhamnosiltransferase BioBrick (Rh1AB_BB)
illegal restriction enzymes lead us to apply multiple site mutagenesis technique.
P. aeruginosa from INDICASAT and gene sequence reviewed in genbank.
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 part is Beta-Agarase cloned out of Pseudoalteromonas atlantica T6C from the ATCC. Pseudoalteromonas atlantica is a gram-negative, motile, chemoorganotroph which lives in sea water. We made a series of primers and cloned agarase using a TOPO cloning kit. Currently the gene has a EcorRI site which limits how it can be assembled.
There is a EcoRI site in the sequence which must be mutated at some point.
Cloned out of Pseudoalteromonas atlantica T6C from the ATCC.
rbs+glgC16 (glgC with G336D substitution)
This is the coding sequence of ''glgC'' (ADP-glucose pyrophosphorylase) from ''Escherichia coli'' JM109 with the substitution G336D, with added ribosome binding site (rbs). This mutation is known to cause increased activity of ADP-glucose pyrophosphorylase in the absence of the activator fructose 1,6-bisphosphate (FBP), high affinity for FBP and substrates lower affinity for the inhibitor AMP. (Leung ''et al.'', 1986; Meyer ''et al.'', 1988)
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.
Limonene synthase gene with ribosome binding site
Ribosome binding site attached to Limonene synthase gene. Limonene synthase enzyme produces (+)-limonene from farnesyl diphosphate (intermediate in mevlonate pathway). (+) Limonene is a component of lemon scent.
several codons were altered silently to remove several forbidden restriction sites. The N-terminal region (plastid targeting sequence) was removed, removing the first 51 amino acids and replacing codon 52 with the start codon ATG. LIMS cDNA was used as genomic DNA contains introns.
Monoterpene biosynthesis in lemon (Citrus limon) cDNA isolation and functional analysis of four monoterpene synthases, J Lucker, et al, Eur. J. Biochem. 269: 3160???3171 (2002) Analysis of Lemon and Bergamot Essential Oils by HPLC with Microbore Columns, M Benincasa, et al, Chromatographia 30(5/6):271 (1990) Citrus fruit flavour and aroma biosynthesis, L Sharon-Asa, The Plant Journal, 36:664 (2003)
metK (from Streptomyces avermitilis, increasing avermectin production)
This part is assembled with geranyl diphosphate synthase (BBa_K727005) and geraniol synthase (BBa_K727006) in order to produce geraniol which generates rose scent. Geraniol is a monoterpenoid and an alcohol. It???s molecular formula is C10H18O . It is the primary part of rose oil, palmarosa oil, and citronella oil (Java type). It also occurs in small quantities in geranium, lemon, and many other essential oils. It has a rose-like odor and is commonly used in perfumes. It is used in flavors such as peach, raspberry, grapefruit, red apple, plum, lime, orange, lemon, watermelon, pineapple, and blueberry.
pcyA converts biliverdin IXalpha (BV) to phycocyanobilin (PCB). Strong RBS (efficiency=0.6).
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.
This is the coding sequence of ''glgC'' (ADP-glucose pyrophosphorylase) from ''Escherichia coli'' JM109 with the substitution G336D. This mutation is known to cause increased activity of ADP-glucose pyrophosphorylase in the absence of the activator fructose 1,6-bisphosphate (FBP), high affinity for FBP and substrates lower affinity for the inhibitor AMP. (Leung ''et al'', 1986; Meyer ''et al.'', 1988)
The mutation G336D was achieved by the substitution of A for G at position 1076 (codon GGC->GAC) by MABEL.
K1321200+J04450 (Vitreoscilla hemoglobin + mRFP)
To measure the characters of Vitreoscilla haemoglobin generator constructed by iGEM14_Imperial (BBa_K1321200). Vitreoscilla haemoglobin can promote the cell growth and protein production ability. We ligate mRFP generator with VHb generator to test the protein production ability in E. coli.
The Vitreoscilla haemoglobin gene sequence in iGEM database is not as the same as that in Genebank but it works.
Naoki Saito,Daiki Haraguchi,Yoshiharu Otaki,Ryuhei Minei
sam8 (tyrosine-ammonia lyase) coding sequence
This is the coding sequence of sam8, encoding tyrosine ammonia lyase, from the bacterium Saccharothrix espanaensis DSM 4429. The enzyme converts tyrosine to p-coumaric acid. Accession: ABC88666. Reference: Berner, M., Krug, D., Bihlmaier, C., Vente, A., Muller, R. and Bechthold, A. 'Genes and Enzymes Involved in Caffeic Acid Biosynthesis in the Actinomycete Saccharothrix espanaensis' J. Bacteriol. 188 (7), 2666-2673 (2006)
Saccharothrix espanaensis DSM 4429. Accession: ABC88666.
pabA is a gene involved in para-aminobenzoic acid (pABA) synthesis from chorismate. PABA is necessary for the synthesis of folate.
We decided to clone the pABA synthesis genes separately (even though they reside side by side) because L.lactis uses start codons (ttg) that E.coli cannot, and so we need to change the start codon for pabB to atg.
This part is an intermediate to the protein generator BBa_45400.
Curdlan synthase gene with codon optimisation for E. coli
The curdlan synthase (73 kDa) coded by crdS (1,965 bp) polymerizes UDP glucose into beta 1,3 D Glucan. CrdS shares no homology with the (1→3)- β-glucan synthase-related FSK1 and FSK2 proteins from fungi (e.g. Saccharomyces, Candida, Aspergillus; Dijkgraaf et al. 2001) or the plant callose synthase-related proteins (Li et al. 2003), which are both classified as GT48 glycosyltransferases. In Agrobacterium, CrdS is an integral inner membrane protein with seven transmembrane (TM) helices, one non-membrane-spanning amphipathic helix and a Nout???Cin disposition). In Agrobacterium, this gene is flancked by two other genes (crdA and crdC) who's corresponding proteins help transport the nascent polymer across the cytoplasmic membrane and into the periplasm. The process of polymerisation mediated by CrdS occurs on the cytoplasmic face of the inner membrane and so is unlikely to involve the periplasmic CrdC, or CrdA directly.
The gene sequence that was used was found in the publication written below. We then verified that the EcoRI, XbaI, SpeI, PstI sequences were not in the gene which was the case. Finally we optimized the sequence using IDT's optimization software on the internet for E.coli. GenBank : AF057142.2 Publication : ????Detection of two loci involved in (1→3)-β-glucan (curdlan) biosynthesis by Agrobacterium sp. ATCC31749, and comparative sequence analysis of the putative curdlan synthase gene???? , Stan J.Stasinopoulos & all, 1999
Curdlan and other bacterial (1→3)-β-D-glucans. M. McIntosh . B. A. Stone . V. A. Stanisich - Appl Microbiol Biotechnol (2005) 68: 163???173; DOI 10.1007/s00253-005-1959-5 (Karnezis et al. 2000, 2003)
rbs-ThpD-TT will produce hydoxyectoine to withstand freeze dry.
Digest I716338 (EcoRI/BamHI, 2057bp, 923bp, S) Sub into Sub into pBca9145-Bca1092 (EcoRI/BglII 2198bp, 9bp, L) Product is I716340
rbs-thpD is the gene that will produce hydroxyectoine to withstand freeze dry.
Digest pBca9145-BBa I716334 (BglII/XhoI, 2063bp, 900bp, S) Sub into pJD507-pSB1A2C (BamHI/XhoI, 2071bp, 9bp, L) Product is I716338
MMT (monomethylamine transferase) catalyses the first reaction of TMA (trimethylamine) synthesis. TMA is the major cause of unpleasant fish odor. For additional information: http://2012hs.igem.org/Team:AUC_Turkey/Project/TMA
α-ALS gene placed under the control of Lux pR promoter. After co-transforming with <bbpart>BBa_K1072000 </bbpart> into E.coli cells,the luxR and AHL complex will activate the Lux pR promotor and transcript α-ALS and GFP genes.
rbs-ThpC will help initiate the production of hydroxyectoine to withstand freeze dry.
Digest pBca9145-BBa I716333 (BglII/XhoI, 2063bp, 405bp, S) Sub into pJD507-pSB1A2C (BamHI/XhoI, 2071bp, 9bp, L) Product is I716337
This Composite Biobrick is created by standard assembly of Part BBa_K118014 (CrtE with rbs), Part BBa_K118006 (CrtB with rbs), Part BBa_K118005 (CrtI with rbs) and Part BBa_K118013 (CrtY with rbs), which are submitted by previous iGEM teams. The whole cassette is on plasmid pSB1A2 (high copy, Ampicillin resistance). Together, enzymes CrtE, CrtB, CrtI and CrtY convert colourless farnesyl pyrophosphate to orange beta-carotene (via intermediates geranylgeranyl pyroiphosphate, phytoene and lycopene). The orange beta-carotene pigment can then be used as a coloured signal output, e.g. for biosensors. There is already individual ribosome binding site before each enzyme gene sequence. Internal restriction sites have been removed by previous iGEM teams. Please refer to Parts BBa_K118014, BBa_K118006, BBa_K118005 and BBa_K118013 for more information on individual Biobricks components. This Composite Biobrick has been put under constitutive promoter R0011 (see Part BBa_K274210) and arabinose-inducible promoter I0500 (see Part BBa_274220), transformed and tested in E.coli strain MG1655. Amount of beta-carotene produced can be measured by photospectrometer with absorbance at 455nm (beta-carotene extraction using acetone). A related Composite Biobrick is Part BBa_K274100 (CrtEBI with rbs), which "stops" at one step before beta-carotene, producing (and retaining) red lycopene pigment.
The colourless precursor, farnesyl pyrophosphate, is naturally produced in E.coli using Non-mevalonate Pathway (from pyruvate and glyceraldehyde-3-phosphate). Our experiences showed that this Biobrick works better in E.coli strains with up-regulated level of precursors. E.coli strain MG1655 demonostrates higher level of pigment (beta-carotene) production than strain TOP10.
Gene coding sequences come from Pantoea ananatis (formerly Erwinia uredovora) (Accession number D90087), a type of Gram negative Enterobacteriaceae. This Composite Biobrick is created by standard assembly of Part BBa_K118014, Part BBa_K118006, Part BBa_K118005 and Part BBa_K118013.
CqsA gene from Vibrio cholerae, codes for the CAI-1 autoinducer synthase. This autoinducer is specific to V. cholerae and is detected by CqsS.
Codon optimized for expression in E. coli.
Vibrio cholerae genomic sequence, from NCBI database, synthesized by GeneArt.
srlD ORF encoding for sorbitol dehydrogenase
Contains start and stop codon and consensus shine-dalgarno sequence.
When used in conjunction with BisdA this enzyme can be used to degrade Bisphenol A. Activity has previously been recorded in E.coli by Miho Sasaki1 et al. 2005. Miho Sasaki1, Jun-ichi Maki, Ko-ichi Oshiman, Yoshinobu Matsumura1, and Tetsuaki Tsuchido.2005, Biodegradation of bisphenol A by cells and cell lysate from Sphingomonas sp. strain AO1. Biodegredation 16: 449-459
When used with BisdA the degredation of Bisphenol A sharply increases
frr (from Streptomyces avermitilis, increasing avermectin production)
Frr gene encodes the ribosome recycling factor (RRF), which is involved in the release of ribosomes from the translational post-termination complex for a new round of initiation. In comparison to the wild-type strain, avermectin yield was increased significantly by 3- to 3.7-fold in transformants 31267(pFRR-1139) and 31267(pFRRerm-1139), both of which contained multiple frr copies, compared with that in the control strains. The avermectin productivity of each culture was quantitatively measured by HPLC analysis. Research indicated that frr overexpression promoted cell growth as well as the expression of ave genes (including pathway-specific positive regulatory gene aveR for avermectin biosynthesis and ave structural genes), leading in turn to avermectin overproduction. Different from S-adenosylmethionine synthetase gene (metK), frr gene revealed a ??????copy number effect??????: multiple copies of frr had a greater promoting effect on avermectin production than a single copy in each of the three transformed S. avermitilis strains.
The metK gene was amplified by PCR with genomic DNA of S. avermitilis(具体型号) as template using primers
COMT (caffeic acid-O-methyl transferase) with ribosome binding site. Enzyme methylates -OH on C4 of the caffeic acid aromatic ring to produce ferulic acid. COMT is the third gene in the vanillin biosynthesis pathway,
Used COMT cDNA as genomic DNA contains introns
Genes and Enzymes Involved in Caffeic Acid Biosynthesis in the Actinomycete Saccharothrix esanaensis, Berner M, et al, Journal of Bacteriology 188(7): 2666, (2006) Gowri G, et al, Molecular cloning and expression of alfalfa S-adenosyl-L-methionine: caffeic acid 3-0-methyltransferase, a key enzyme of lignin biosynthesis, Plant Physiology 97(1):7 (1991) Downregulation of Caffeic Acid 3-O-Methyltransferase and Caffeoyl CoA 3-O-Methyltransferase in Transgenic Alfalfa Impacts on Lignin Structure and Implications for the Biosynthesis of G and S Lignin, Guo D, et al, Plant Cell. January; 13(1): 73 (2001)
This version of the LuxC comes from Vibrio salmonicida. Team Example used it to build a bacterial lava lamp.
No modifications were necessary in order to comply with the standard.
Vibrio salmonicida hypothetical protein EJN1 gene, complete cds; luminescence gene cluster, complete sequence; and RibG (ribG) gene, complete cds. NCBI Accession number is AF452135 REGION: 2346..3776
sam5 (coumarate hydroxylase) with native rbs.
Sam5 (4-coumarate-3-hydroxylase) of the bacterium Saccharothrix espanaensis DSM 44229 with its native rbs.
This was made as a single biobrick, rather than separate rbs and coding sequence, hence the use of blunt ligation in generating this composite part.
This part encodes two enzymes, rhlA and rhlB (BBa_K653000), for biosynthesis of rhamnolipid. The enzymes are under T7 promoter with medium strengh and a strong ribosomal binding site (BBa_K613010), and stopped with a double terminator (BBa_B0015). The promoter had the second highest efficiency as shown by EPFL team 2011 (http://parts.igem.org/Part:BBa_K613010:Experience).
We include the scar sequences between RBS-coding and coding-terminator in hope that it will function properly.
The part was chemically synthesised. The sequences were based on parts available in Registry.
luxI protein synthesizes 3OC6HSL, which binds to LuxR. Strong RBS (efficiency=0.6).
Similarly to OtsA and OtsB, rbs-ThpB will help initiate the production of hydroxyectoine to withstand freeze dry.
Digest I716332 (BglII/XhoI, 2063bp, 1269bp, S) Sub into pJD507-pSB1A2C (BamHI/XhoI, 2071bp, 9bp, L) Product is I716336
This part is assembled with Geranyl Diphosphate Synthase (http://partsregistry.org/wiki/index.php?title=Part:BBa_K727005) and Geraniol synthase (BBa_K727006)in order to produce geraniol. Geraniol is a monoterpenoid and an alcohol. It???s molecular formula is C10H18O . It is the primary part of rose oil, palmarosa oil, and citronella oil (Java type). It also occurs in small quantities in geranium, lemon, and many other essential oils. It has a rose-like odor and is commonly used in perfumes. It is used in flavors such as peach, raspberry, grapefruit, red apple, plum, lime, orange, lemon, watermelon, pineapple, and blueberry.
sam5 (coumarate hydroxylase) coding sequence
This is the coding sequence of sam5, encoding 4-coumarate-3-hydroxylase from the bacterium Saccharothrix espanaensis. The enzyme converts p-coumaric acid to caffeic acid. Accession: ABC88666. Reference: Berner, M., Krug, D., Bihlmaier, C., Vente, A., Muller, R. and Bechthold, A. 'Genes and Enzymes Involved in Caffeic Acid Biosynthesis in the Actinomycete Saccharothrix espanaensis' J. Bacteriol. 188 (7), 2666-2673 (2006). Note: contains a PstI site. This will be removed by site directed mutagenesis in due course but is still present, near the 3' end, in this version.
Note that this version of the gene contains a PstI site near the 5' end. This will ultimately be removed by site directed mutagenesis.
Saccharothrix espanaensis DSM 44229, sequence accession ABC88666.
Simple inducible (LacI) ethylene generator
A simpler version of BBa_K417006 which expresses ACC synthase and ACC oxidase (EFE) to produce ethylene. This version does not express the SAM synthetase enzyme (BBa_K417000) and relies on S-adenosyl methionine generated by the chassis.
Native LacI promoter (BBa_R0010) was chosen since it facilitates easier assembly compared with the shorter alternative BBa_R0011, although it would be worth testing both these promoters. It is unknown if the order of the coding sequences is important in this device.
See part documentation for enzyme coding sequences BBa_K417001 and BBa_K417002 for details on the origin of the sequences.
Cytochrome A and X from Nitrosomonas europaea
Denitrification operon from Pseudomonas aeruginosa
Kristian Davidsen, Helen Cook and Jakob Berg Jespersen
rbs+glgC (ADP-glucose pyrophosphorylase)
This is the coding sequence of ''glgC'' from ''Escherichia coli'' JM109 with added ribosome binding site (rbs). It encodes ADP-glucose pyrophosphorylase, which catalyses Glucose 1-phosphate + ATP -> ADP-glucose + PPi, a step in the production of glycogen from glucose.
This Composite Biobrick is created by standard assembly of Part BBa_K118014 (CrtE with rbs), Part BBa_K118006 (CrtB with rbs) and Part BBa_K118005 (CrtI with rbs), which are submitted by previous iGEM teams. The whole cassette is on plasmid pSB1A2 (high copy, Ampicillin resistance). Together, enzymes CrtE, CrtB and CrtI convert colourless farnesyl pyrophosphate to red lycopene (via intermediates geranylgeranyl pyroiphosphate and phytoene). The red lycopene pigment can then be used as a coloured signal output, e.g. for biosensors. There is already individual ribosome binding site before each enzyme gene sequence. Internal restriction sites have been removed by previous iGEM teams. Please refer to Parts BBa_K118014, BBa_K118006 and BBa_K118005 for more information on individual Biobricks components. This Composite Biobrick has been put under constitutive promoter R0011 (see Part BBa_K274110) and arabinose-inducible promoter I0500 (see Part BBa_274120), transformed and tested in E.coli strain MG1655. Amount of lycopene produced can be measured by photospectrometer with absorbance at 475nm (lycopene extraction using acetone).
The colourless precursor, farnesyl pyrophosphate, is naturally produced in E.coli using Non-mevalonate Pathway (from pyruvate and glyceraldehyde-3-phosphate). Our experiences showed that the Composite Biobrick, when put under a promoter, works better in E.coli strains with up-regulated level of precursors. E.coli strain MG1655 demonostrates higher level of pigment (lycopene) production than strain TOP10.
Gene coding sequences come from Pantoea ananatis (formerly Erwinia uredovora) (Accession number D90087), a type of Gram negative Enterobacteriaceae. This Composite Biobrick is created by standard assembly of Part BBa_K118014, Part BBa_K118006 and Part BBa_K118005.
sam8 (tyrosine ammonia lyase) with native rbs
This is sam8 (tyrosine ammonia lyase) of the bacterium Saccharothrix espanaensis DSM 44229 with its native ribosome binding site.
This part was made as a single biobrick rather than separate rbs and coding sequence, hence the use of blunt ligation in generating the final sequence.
Crystal protein gene (cry11Aa) from Bacillus thuringiensis subsp. Israelensis.
The cry weapon system produce crystal protein, targetting the wrigglers, larvae of mosquitoes. The cry11Aa gene is cloned from Bacillus thuringiensis subsp. Israelensis. The cry protein is controlled by the tetR-repressible promoter PtetR (BBa_R0040), which in turn is regulated by a temperature control system. When E.coli is released into environment (<37C), tetR begins to degrade, resulting in the promoter PtetR (BBa_R0011) expressing downstream genes, cry11Aa (BBa_K332012) and Green Fluorescent Protein (GFP)(BBa_E0040).
Procedures (I) Culture Bti. Bacillus thuringiensis subsp. Israelensis (BCRC15860) 1. Prepare agar plate for Bti. Beef extract 3.0g Peptone 5.0g Agar 15.0g ddH2O 1 L *adjust PH to 7.0 *No antibiotic *Be aware of contamination 2. Plate Bti. on the medium and incubate the cultures at 30??C overnight. (II) Clone cry11Aa from Bti. into TA vector 1. Extract genomic DNA of Bti. by liquid nitrogen. 2. Add some ddH2O to dilute the DNA. 3. Design primers Vf2: ATTCAATAAAAGGTGGAATGAATTATGGA Tm: 53??C VR: GTGCTAACATGACTTCTACTTTAGT Tm: 52.8??C 4. Find the best PCR condition by gradient PCR. Anneling temperature: 51??C??10??C 5. PCR by B-taq plus on the best condition Template DNA（10ng/μl） 2.0 B-taq buffer 5.0 dNTP（2mM） 5.0 forward primer（10μM） 1.5 reverse primer（10μM） 1.5 B-taq plus DNA polymerase（2Kb） 1.0 ddH2O 34.0 Total 50 μl 6. Digestion to confirm the cry11Aa fragment and enzyme sites. 7. TA clone 8. DNA sequencing (III) PCR mutagenesis at two enzyme sites --- EcoR1 and Spe1 1. Design primers by primerX. EcoR1-Vf2: CGG GTA CAA TCT CAG AAC TCG GGA AAT AAT AGA A EcoR1-VR: TTC TAT TAT TTC CCG AGT TCT GAG ATT GTA CCC G Spe1-Vf2: ATA ATG AAT GGG GAG GAC TGG TTT ATA AGT TAT TAA TGG G Spe1-VR: CTT CCC CCA TTA ATA ACT TAT AAA CTA GTC CTC CCC ATT CAT 2. Digestion to confirm the fragments (IV) PCR construction of Biobrick parts 1. Design primers by Assembly standard 10. Vf2: GTTTCTTC GAATTC GCGGCCGC T TCTAG ATGGAAGATAGTTCTTTAGATACT VR: GTTTCTTC CTGCAG CGGCCGC T ACTAGT A CTACTTTAGTAACGGATT 2. PCR condition 3. Ligation to backbones(Psb1C3). (V) Transform into E.coli 1. Thaw competent cells and BBa_K332012 plasmid on ice. 2. Add 2ul plasmid to competent cell and place in ice for 5 minutes. 3. Put the transformed cells into 42℃ water bath for 45 seconds. 4. Plate the cells on the appropriate media and antibiotic, such as agar plates with 25 ??g/ml kanamycin. 5. Incubate the cultures at 37??C overnight. (VI) Culture with mosquito larvae and observe
Crystal protein gene (cry11Aa) from Bacillus thuringiensis subsp. Israelensis. BCRC Number: 15860 Organism: Bacillus thuringiensis subsp. israelensis de Barjac History: << ATCC << USDA << ONR 60 << H. de Barjac << L. J. Goldberg 60 Other Collection No.: ATCC 35646 Source: sewage, Israel Characterization: Production of delta-endotoxin against dipteran larvae --B3009 Others: referred to as the type strain --B3008 Growth Conditions: 30??C , Medium ID: 3 Oxygen Requirement: Aerobic Biosafety Level: 1 References: Temeyer, K. B. 1984. Larvicidal activity of Bacillus thuringiensis subsp. israelensis in the dipteran Haematobia irritans. Appl. Environ. Microbiol.47 : 952-955. PMID: 6742837. Demezas, D. H. and J. Bell. 1995. Evaluation of low molecular weight RNA profiles and ribotyping to differentiate some Bacillus species. Syst. Appl. Microbiol.18 : 582-589.
When used in conjunction with BisdB this enzyme can be used to degrade Bisphenol A. Activity has previously been recorded in E.coli by Miho Sasaki1 et al. 2005. Miho Sasaki1, Jun-ichi Maki, Ko-ichi Oshiman, Yoshinobu Matsumura1, and Tetsuaki Tsuchido.2005, Biodegradation of bisphenol A by cells and cell lysate from Sphingomonas sp. strain AO1. Biodegredation 16: 449-459
These parts where designed to include a his tag to make it easier to view enzyme presence
lasI: autoinducer synthetase for PAI from Pseudomonas aeruginosa. Strong RBS (efficiency=0.6).
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.
glgC coding sequence encoding ADP-glucose pyrophosphorylase
This is the coding sequence of ''glgC'' from ''Escherichia coli'' JM109. It encodes ADP-glucose pyrophosphorylase, which catalyses Glucose 1-phosphate + ATP -> ADP-glucose + PPi, a step in the production of glycogen from glucose.
The genomic DNA sequence contains to EcoRI sites, both of which have been mutated out using the mutagenesis by blunt-ended ligation (MABEL) technique