We have designed a tunable microbial consortium to distribute the 4S biodesulfurization pathway, responsible for the bio-desulfurization of DBT, as a metabolic network.
There are two major genetic circuits contained within our system:
The first is responsible for tuning the relative populations of the bacteria within the consortium. It is composed of different fluorescence markers under constitutive promoters, used to differentiate member of the population. As well as an amino acid biosynthesis genes under a inducible promoter, used to regulate the bacterial populations within the consortium.
The second codes for the distributed 4S pathway, and was created by splitting the dsz operon into each member species of our tunable consortium.
Data for our Favourite New PartsMain Page - Strong Constitutive Promoter-ECFP generator, BBa_K804000: This is an Enhanced Cyan Fluorescence Protein under a strong constitutive Ptet promoter (BBa_J23118). It constitutively expresses ECFP (BBa_E0420). The CFP output device does not have a LVA tag and has a strong RBS. Under a plate scanner, ECFP excites at 439nm and emits at 476nm. The fluorescence output from this construct can be used to monitor growth and population dynamics(only at exponential phase) in a microbial consortium.
Main Page - Strong constitutive promoter-EYFP generator, BBa_K804001: This is an Enhanced Yellow Fluorescence Protein under a strong constitutive Ptet promoter (BBa_J23118). It constitutively expresses EYFP (BBa_E0430). The CFP output device does not have a LVA tag and has a strong RBS. Under a plate scanner, EYFP excites at 514nm and emits at 527nm. The fluorescence output from this construct can be used to monitor growth and population dynamics(only at exponential phase) in a microbial consortium.
Main Page - Rhamnose Inducible TrpA coding gene, BBa_K804012: This part contains a rhamnose inducible (pRha) TrpA coding gene. Upon induction with rhamnose, TrpA is expressed for indole-3-glycerol-phosphate(InGP)binding and catalysis/cleavage of InGP to indole and glyceraldehyde-3-phosphate during an α reaction. This construct can be used to induce growth in Trp- auxotrophs.
Data for Pre-Existing PartsExperience - Rhamnose inducible, glucose repressible promoter (pRha), BBa_K902065 (Calgary, iGEM 2012): We placed the Rhamnose promoter upstream of our TrpA and MetA coding genes. In the respective auxotrophs, induction by arabinose resulted in growth compared to a negative control as measured by a plate reader.
Experience - Enhanced yellow fluorescent protein, BBa_E0030 (Registry, iGEM 2004): We placed the fluorescent protein gene downstream of a strong constitutive promoter (New Favourite Part BBa_K804001) and measured fluorescence and OD by a plate reader.
Experience - Engineered cyan fluorescent protein, BBa_E0020 (Registry, iGEM 2004): We placed the fluorescent protein gene downstream of a strong constitutive promoter (New Favourite Part BBa_K804000) and measured fluorescence and OD by a plate reader.
We've also characterized the following partsMain Page - Constitutive ECFP generator - Arabinose inducible TrpA coding gene, BBa_K804007: This part contains a constitutive ECFP generator along with an arabinose inducible (Pbad) Trp A coding gene. Upon induction with arabinose, TrpA is expressed for indole-3-glycerol-phosphate(InGP)binding and catalysis/cleavage of InGP to indole and glyceraldehyde-3-phosphate during an α reaction. This construct can be used to induce growth in Trp- auxotrophs and can be monitored by fluorescence for its population dynamics in co-culture with other auxotrophs.
Main Page - Constitutive EYFP generator - Arabinose inducible TrpA coding gene, BBa_K804009: This part contains a constitutive EYFP generator along with an arabinose inducible (Pbad) Trp A coding gene. Upon induction with arabinose, TrpA is expressed for indole-3-glycerol-phosphate(InGP)binding and catalysis/cleavage of InGP to indole and glyceraldehyde-3-phosphate during an α reaction. This construct can be used to induce growth in Trp- auxotrophs and can be monitored by fluorescence for its population dynamics in co-culture with other auxotrophs.
Main Page - Constitutive EYFP generator - Arabinose inducible MetA coding gene, BBa_K804011: This part contains a constitutive EYFP generator along with an arabinose inducible (Pbad) Met A coding gene. Upon induction with arabinose, MetA is expressed for for the biosynthesis of de novo methionine. It encodes for the MetA enzyme which transfers a succinyl group from succinyl-CoA to homoserine, forming O-succinyl-L-homoserine, a precursor of methionine. This construct can be used to induce growth in Met- auxotrophs and can be monitored by fluorescence for its population dynamics in co-culture with other auxotrophs.
Main Page - TrpA coding gene, BBa_K804002: This is the TrpA coding gene. It codes for the alpha subunit of tryptophan synthase (TSase α), and functions as both a binding site for indole-3-glycerol-phosphate (InGP) and can catalyze the cleavage of InGP to indole and glyceraldehyde-3-phosphate. This is the same part as BBa_K187028, but is in the pSB1C3 plasmid.
Main Page - TyrA coding gene, BBa_K804003: This is a TyrA coding gene for both the tyrosine and phenylalanine bio-synthetic pathways. TyrA expresses a bifunctional chorismate mutase/prehenate dehydrogenase which catalyzes the conversion of chorismate into prephenate and NAD+-dependent oxidative decarboxylation of prephanate.
Main Page - MetA coding gene, BBa_K804004: This is a MetA coding gene for the biosynthesis of de novo methionine. It encodes for the MetA enzyme which transfers a succinyl group from succinyl-CoA to homoserine, forming O-succinyl-L-homoserine, a precursor of methionine.
Main Page - DszC coding gene, BBa_K804005: This is a DszC coding gene from the DszABC operon in the 4S pathway of Rhodococcus erythropolis IGTS8. It encodes one of the three biodesulfurizing enzymes in the DszABC operon. DszC enzymes have been shown to catalyze the oxidation of dibenzothiophene (DBT)to dibenzothiophene-5-oxide (DBTO) in the first reaction and then from DBTO to DBT sulfoxide (DBTO2) in the second reaction, both in the presence of NADH, oxygen, FMN and flavin reductases.
Main Page - DszD coding gene, BBa_K804006: This is a DszD coding gene isolated from Rhodococcus erythropolis IGTS8, which encodes for a NADH:FMN oxidoreductase to enhance the activities of DszA and DszC in the DszABC operon.
Main Page - Arabinose inducible TrpA coding gene, BBa_K804008: This part contains an arabinose inducible (Pbad) Trp A coding gene. Upon induction with arabinose, TrpA is expressed for indole-3-glycerol-phosphate(InGP)binding and catalysis/cleavage of InGP to indole and glyceraldehyde-3-phosphate during an α reaction. This construct can be used to induce growth in Trp- auxotrophs.
Main Page - Arabinose inducible MetA coding gene, BBa_K804010: This part contains an arabinose inducible (Pbad) Met A coding gene. Upon induction with arabinose, MetA is expressed for for the biosynthesis of de novo methionine. It encodes for the MetA enzyme which transfers a succinyl group from succinyl-CoA to homoserine, forming O-succinyl-L-homoserine, a precursor of methionine. This construct can be used to induce growth in Met- auxotrophs.
Main Page - Rhamnose Inducible MetA coding gene, BBa_K804013: This part contains a rhamnose inducible (pRha) MetA coding gene. Upon induction with rhamnose, MetA is expressed for for the biosynthesis of de novo methionine. It encodes for the MetA enzyme which transfers a succinyl group from succinyl-CoA to homoserine, forming O-succinyl-L-homoserine, a precursor of methionine. This construct can be used to induce growth in Met- auxotrophs.
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