isochorismate pyruvate-lyase and isochorismate synthase (pchBA); converts chorismate to salicylate
pchBA codes for proteins used to generate salicylic acid. It doesn't make a fusion protein, even though the coding regions overlap; the RBS for pchA is located at the end of the coding region of pchB.
pchBA is found in pseudomonas. The plasmid was kindly sent to the MIT iGEM team by Dr. Cornelia Riemmann from the Universite de Lausanne.
This part is an intermediate to the protein generator BBa_45220. This part is almost identical to BBa_J45199 except that it uses B0032 as its RBS (instead of B0030).
Protein generator converting PoPS to the protein ATF1. ATFI encodes the enzyme alcohol acetyltransferase I (AATase I), which produces acetate esters from acetyl CoA and alcohols. This part is similar to BBa_J45200, except that it uses the B0032 RBS instead of B0030 (which is in BBa_J45200).
This part is the ATF sequence BBa_45J006 with a single point mutation at base pair 1148 (T -> C). Despite the mutation, the enzyme is still functional (kinetic studies have not been done to determine realitive functionality).
SAM:benzoic acid/salicylic acid carboxyl methyltransferase I; converts salicylic acid to methyl sali
Codes for BSMT enzyme to convert Benzoic acid/salicylic acid to methyl benzoate and methyl salicylate. This part is phBSMT1 from Petunia x hybrida.
Species: Petunia x hybrida from Genbank; (also available: A. thaliana and N. suaveolens)
Wintergreen odor enzyme (BSMT1) generator
This is the wintergreen generating device hooked up to a constitutive promoter.
This is made from a biobrick promoter and our biobrick wintergreen generating device.
This composite part include a ribosome binding site, the BSMT coding region, and a terminator. This part is attached to different terminators.
We used the earlier composite biobrick part of the BSMT+Terminator and attached a RBS from the registry.
T7 promotor (under the control of lacI) and downstream CecropinXJ
Antimicrobial peptide CecropinXJ belongs to AMP family Cecropin, a group of small basic polypeptides mainly found in the hemolymph of insects, consist of 31-39 amino acid residues and have a broad spectrum, high heat stability and potent bacteriostatic activity.
The coding sequence was optimized for E.coli expression using JCat http://www.jcat.de/. The single stop codon was replaced by double TAA stop codon.
The original sequence of CecropinXJ comes from GenBank NM_001043995.1. The sample used for experiments was artificially synthesized by Tsingke.
Stationary-phase-dependent wintergreen odor generator
This is the wintergreen generating device hooked up to a stationary phase promoter with RBS B0032.
Biobricks of osmY promoter and intermediate 45119.
Still in development as SPP is not confirmed to work.
Exponential phase dependent wintergreen odor generator
This is a protein generator that outputs BSMT (benzoic acid/salicylic acid methyl transferase) under control of the osmY stationary phase promoter and Q04400 inverter. Ideally, when in stationary phase, BSMT output is "low" and vice versa.
Exponential phase wintergreen odor biosynthetic system
branched-chain amino acid transaminase (BAT2); converts leucine to alpha-ketoisocaproate
Stationary phase banana odor biosynthetic system
This system designed by the MIT 2006 iGEM team generates mint smell by biosynthesizing salicylic acid from chassis-produced metabolites (chorismate) and then converts salicylic acid into methyl salicylate (wintergreen scent).
We recommend use of strain <bbpart>BBa_J45999</bbpart> as it does not produce indole. Indole is responsible for the foul smell of wt e.coli, and masks the wintergreen scent produced by J45600.
This system is composed of devices <bbpart>J45120</bbpart> and <bbpart>J45320</bbpart>.
This part has EcoRI and SpeI sites in it. Use caution when conducting standard assembly with the part.
This part was made via standard assembly of J45200 and J45400.
SAM:salicylic acid carboxyl methyltransferase; converts salicylic acid to methyl salicylate (winter
SAM:salicylic acid carboxyl methyltransferase; converts salicylic acid to methyl salicylate (winterg
we need an MTA or to sequence the coding region for this enzyme
Constitutive promotor J23105 and downstream CecropinXJ
Biobrick BBa_K1919001 is a composite part, consisting of constitutive promotor J23105, RBS, TrxA tag, 6xHis tag, thrombin site, S tag, enterkinase site and the coding sequence of antimicrobial peptide CecropinXJ.
Two steps were needed in order to obtain BBa_K1919001. Step 1, BBa_K1919000 was inserted into pET32a(+). The primers used for PCR are as following: F: 5'-CCGGGATCCATGAACTTCGCTAAAATCCTGTCTT-3' (BamH I site was added) R: 5'-CCGCTCGAGTTATTATTTACCGATAGCTTTA-3' (Xho I site was added) BamH I/Xho I Double enzymatic cutting was used for both pET32a(+) and BBa_K1919000. Step 2, RBS and 5 downstream tags as well as original CecropinXJ was inserted at the downstream of promotor J23105 with the same procedure as step 1. The primers used for PCR are as following: F: 5'-GCTCTAGAGCGAAGGAGATATACATATGAGCG-3' (Xba I site was added) R: 5'-TGCACTGCAGTGCAGACTAGTCGTTATTGCTCAGCGGTGG-3'(Pst I site was added) Xba I/Pst I double enzymatic cutting was used for PCR product and Spe I/Pst I double enzymatic cutting was used for BBa_J23105. After enzymatic linkage, the construction of BBa_K1919001 was finished. Then this part was added to pSB1C3 backbone.
The sequence of constitutive promotor J23105 comes from biobrick BBa_J23105. RBS and 5 downstream tags come from commercial plasmid pET32a(+). The sequence of CecropinXJ comes from biobrick BBa_K1919000.
This system designed by the MIT 2006 iGEM team and generates mint smell by biosynthesizing salicylic acid from chassis-produced metabolites (chorismate) and then converts salicylic acid into methyl salicylate (wintergreen scent).
alcohol acetyltransferase I; converts isoamyl alcohol to isoamyl acetate (banana odor)
This part is the ATF sequence (BBa_45J006) with a single point mutation at base pair 1176 (T -> C).
It has a single point mutation at base pair 1176 (T -> C).
SAM:jasmonic acid carboxyl methyltransferase; converts jasmonic acid to jasmine odor
This ORF encodes the enzyme responsible for catalyzing the following reaction: Jasmonic Acid--->Methyl Jasmonate It is initially found in ''Arabidopsis thaliana'' but has been transferred successfully to the E. coli genome by Seo, et al. The product, methyl jasmonate has a jasmine smell to it.
Km: 38.5 ??M; Kcat:25 S−1 for Jasmonic Acid--->Methyl Jasmonate with the aid of JAMT: Jasmonic acid carboxyl methyltransferase
If the part becomes physically available, it would probably be donated by Seo, et al.
E. coli strain YYC912; Odor-free chassis
Escherichia coli strain YYC912. Biosafety level 1 organism.
This strain was obtained from the E.coli Genetic Resources: CGSC, The Coli Genetic Stock Center http://cgsc.biology.yale.edu/. Strain YYC912.
Wintergreen odor enzyme (BMST1) generator
This composite part include a ribosome binding site (B0032 different from J45099), the BSMT coding region, and a terminator. This part is attached to different promoters.
Derived from the early part J45098 and sister to J45099.
SAM:benzoic acid carboxyl methyltransferase; converts benzoic acid to methyl benzoate (floral odor)
BAMT catalyzes the transfer of a methyl group from SAM (S-adenosyl methyltransferase) to benzoic acid, creating methyl benzoate. Methyl benzoate produces a "pleasant" smell.
Genbank accession number(s): AF198492 This part is from A. majus.
Protein generator converting PoPS to the prot BSMT. The BSMT enzyme converts benzoic acid/salicylic acid to methyl benzoate and methyl salicylate.
This part is phBSMT1 from Petunia x hybrida (GenBank). See part design and discussion for more information.
This is the wintergreen generating device hooked up to a constitutive promoter with a different RBS (B0032, different from 45100).
crtI coding sequence encoding phytoene dehydrogenase
This is the coding sequence of crtI from Pantoea ananatis (formerly Erwinia uredovora) (Accession number D90087). It encodes phytoene dehydrogenase, part of the carotenoid biosynthesis pathway, which converts Phytoene to Lycopene.
The native crtI sequence contains two internal PstI sites. Primers crtImutf1 and crtImutf2 were ordered so that the MABEL protocol (invented for this purpose) could be used to remove these sites. The mutagenic primers were as follows: * crtImutf1: gtt tac agt aag gtt gcc * crtImutf2: agt aac tct ctg ttt gtg c In both cases, the PstI site CTGCAG was converted to CTGCAA, a silent mutation.
This part converts salicylic acid into methyl salicylate (a mint-smelling compound) in the exponential phase of cell growth. The part consists of a stationary phase promoter (J45992), connected to an inverter (Q04401) as well as the methyl salicylate protein generator (J45119). This part replaces the defective part J45180, which used the truncated version of the stationary phase promoter (J45993). This part did not function as it was designed due to an imbalance between the strength of the stationary phase promoter and the strength of the promoter in the inverter (R0040).
We needed the full osmY promoter strength to be relatively comparable to the R0040 promoter strength.
Stationary-phase-dependent banana odor generator
This part is an ATF1-1148 protein generator under control by the osmY stationary phase promoter (BBa_J45993). It is almost identical to J45250, except that: 1. The promoter is the truncated version of osmY and 2. it uses the B0030 RBS instead of B0032 (which is in J45250).
This enzyme catalyzes the reaction of isoamyl alcohol -> isoamyl acetate
This part is an intermediate to the protein generator BBa_45200.
COMT coding sequence Encodes caffeic acid-O-methyl transferase (methylates -OH on C4 of the aromatic ring) from Medicago sativa (alfalfa), synthesised by Geneart
Used cDNA as coding gene in Alfalfa genome contains introns.
Alfalfa cDNA 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 part is an ATF1-1148 protein generator under control by the osmY stationary phase promoter. It is almost identical to J45270, except that: 1) The promoter is the truncated version of osmY and 2) it uses the B0030 RBS instead of B0032 (which is in J45270).
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)
Constitutive promotor J23106 and downstream CecropinXJ
Biobrick BBa_K1919002 is a composite part, consisting of constitutive promotor J23106, RBS, TrxA tag, 6xHis tag, thrombin site, S tag, enterkinase site and the coding sequence of antimicrobial peptide CecropinXJ.
Two steps were needed in order to obtain BBa_K1919006. Step 1, BBa_K1919000 was inserted into pET32a(+). The primers used for PCR are as following: F: 5'-CCGGGATCCATGAACTTCGCTAAAATCCTGTCTT-3' (BamH I site was added) R: 5'-CCGCTCGAGTTATTATTTACCGATAGCTTTA-3' (Xho I site was added) BamH I/Xho I Double enzymatic cutting was applied for both pET32a(+) and BBa_K1919000. Step 2, RBS and 5 downstream tags as well as original CecropinXJ was inserted at the downstream of promotor J23106 with the same procedure as step 1. The primers used for PCR are as following: F: 5'-GCTCTAGAGCGAAGGAGATATACATATGAGCG-3' (Xba I site was added) R: 5'-TGCACTGCAGTGCAGACTAGTCGTTATTGCTCAGCGGTGG-3'(Pst I site was added) Xba I/Pst I double enzymatic cutting was applied for PCR product and Spe I/Pst I double enzymatic cutting was used for BBa_J23106. After enzymatic linkage, the construction of BBa_K1919002 was finished. Then this part was transfered into pSB1C3 backbone.
The sequence of constitutive promotor J23106 comes from biobrick BBa_J23106. RBS and 5 downstream tags come from commercial plasmid pET32a(+). The sequence of CecropinXJ comes from biobrick BBa_K1919000
SAM:benzoic acid/salicylic acid carboxyl methyltransferase II; converts salicylic acid to methyl sal
Codes for BSMT enzyme to convert Benzoic acid/salicylic acid to methyl benzoate and methyl salicylate. This part is phBSMT2 from Petunia x hybrida.
Synthetic periplasmic binding protein that docks a vanillin molecule
Stationary phase dependent wintergreen odor generator
This is the wintergreen generating device hooked up to a stationary phase promoter with RBS B0030.
Biobricks of osmY promoter and intermediate 45099.
ketoisocaproate decarboxylase (THI3); converts alpha-ketoisocaproate to 3-methylbutanal
Protein generator converting PoPS to the protein ATF1. ATFI encodes the enzyme alcohol acetyltransferase I (AATase I), which produces acetate esters from acetyl CoA and alcohols.
This part was cloned from S. cerevisiae.
This part is an intermediate to the protein generator BBa_45400.
Constitutive banana odor enzyme (ATF1) generator
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)