Parts

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Parts CISS iGEM 2025

Parts for E.Coli:

Type Part Link Name Functional Description
Composite (Device) BBa_25D3UKXW PET to TPA & EG using LCCICCG in E.coli Indicuble expression under the T7 promoter of the modified enzyme LCCICCG (leaf-branch compost cutinase) - https://parts.igem.org/Part:BBa_K3478888 GFP is also under the same promoter allowing for measurements of expression and optimization of conditions to maximize enzyme production. Biotechnology applications exist for LCCICCG to convert PET into TPA which can be further metabolised into higher value products (Sadler and Wallace, 2021).
Composite (Device) BBa_2522T7A8 TPA transporter in E.coli TPA transporter under constitutive promoter for E.coli. Contains the tpaK gene (https://parts.igem.org/Part:BBa_K4728011) originating from the species Rhodococcus jostii, tpaK encodes for the terephthalate transporter used during uptake. This transport protein allows for the transportation of terephthalic acid into the bacterial cells. This part can allow for combination with TPA metabolizing enzymes for upcycling of TPA into products such as Vanillin (Sadler and Wallace, 2021 DOI:https://doi.org/10.1039/D1GC00931A) or PCA.
Composite (Device) BBa_25CF2NOP TPA to PCA in E.coli This part allows for the uptake of Terephthalate (TPA) in E.coli and concurrently for the metabolism of TPA into PCA (protocatechuate). This is designed to be an essential step in the biotechnological process of upcycling PET plastic into higher value products. This composite part uses basic parts coding for genes tphA1, 2, 3, and DCDDH which produce enzymes involved in the conversion of TPA to PCA.
Basic BBa_25VDWFRT pcaGH - Protocatechuate 3,4-dioxygenase beta and alpha subunits Pseudomonas putida (ATCC 23975) protocatechuate 3,4-dioxygenase beta and alpha subunits (pcaH and pcaG) genes, complete cds.. Codes for the enzyme Protocatechuate 3,4-dioxygenase, part of the beta-ketoadipate pathway. Catalyzes the production of 3-carboxy-cis,cis-muconate from 3,4-dihydroxybenzoate.
Basic BBa_251AN6OX pcaIJ – beta-ketoadipate: succinyl-coA transferase Pseudomonas putida beta-ketoadipate: succinyl-coA transferase (pcaI, pcaJ) genes, complete cds.
Basic BBa_25JXTXSM pcaB - 3-carboxy-cis,cis-muconate cycloisomerase (pcaB) gene b-Carboxy-cis,cis-muconate lactonizing enzyme (cycloisomerase) from P. putida. Part of the beta-ketoadipate pathway; Facilitating the reaction: 5-oxo-4,5-dihydro-2-furylacetate from 3-carboxy-cis,cis-muconate.
Basic BBa_256JN97U PcaF - Beta-ketoadipyl-CoA thiolase PcaF gene from Pseudomonas putida KT2440. Catalyzes thiolytic cleavage of beta-ketoadipyl-CoA to succinyl-CoA and acetyl-CoA.
Basic BBa_25T1AMVB PCA (protocatechuate) Transporter 4-hydroxybenzoate transporter PcaK from Pseudomonas aeruginosa. Transports 4-hydroxybenzoate (4-HBA) and protocatechuate across the membrane. Driven by the proton motive force (https://www.uniprot.org/uniprotkb/Q9I6Q3/entry).
Composite (Device) BBa_25VAG5KT PCA transporter in E.coli TPA transporter under constitutive promoter for E.coli. Contains the pcaK gene (https://registry.igem.org/parts/bba-25t1amvb) originating from the species Pseudomonas aeruginosa, pcaK transports 4-hydroxybenzoate (4-HBA) and protocatechuate across the membrane. Driven by the proton motive force. (https://www.uniprot.org/uniprotkb/Q9I6Q3/entry)
Composite (Device) BBa_254GSRYG Protocatechuate (PCA) to Acetyl CoA in E.coli Conversion of PCA to Acetyl CoA via several enzymes from the beta-ketoadipate pathway. The system is inducible under the T7 promoter for string expression in E.coli.
Composite (Device) BBa_25GP9EZL PHB production and optimization with ZWF in E.coli For the inducible (T7) production of Poly-3-hydroxybutyrate, (PHB) in E.coli, a type of Polyhydroxyalkanoate (PHA) which is a biodegradable plastic which accumulates inside cells. PHB is produced via the biosynthesis of 3 enzymes encoded by the genes phaA, phaB and phaC. Optimized with the ZWF gene which codes for Glucose-6-phosphate 1-dehydrogenase, which Catalyzes the oxidation of glucose 6-phosphate to 6-phosphogluconolactone. The purpose of this gene is to increase the amount of NADPH available (Lim et al., 2002) and thus upregulate the PHB biosynthesis pathway.
Composite (Device) BBa_25HZF1BX PHB production and optimization with SerA in E.coli For the inducible (T7) production of Poly-3-hydroxybutyrate, (PHB) in E.coli, a type of Polyhydroxyalkanoate (PHA) which is a biodegradable plastic which accumulates inside cells. PHB is produced via the biosynthesis of 3 enzymes encoded by the genes phaA, phaB and phaC. To up-regulate the biosynthesis pathway of PHB production, this part also contains Serine Repeat Antigen (SerA) gene (https://parts.igem.org/Part:BBa_K2086001). This gene codes for the protein 3-phosphoglycerate dehydrogenase, also known as PHGDH. The purpose of this gene is to increase the amount of NADPH available (Zhang et al., 2014) and thus upregulate the PHB biosynthesis pathway.
Composite (Device) BBa_254YY4J3 Polyhydroxybutyrate (PHB) depolymerase under T7 promoter with GFP Under inducible expression via the T7 promoter, the extracellular poly(3-hydroxybutyrate) (PHB) depolymerase PhaZ - isolated from Penicillium funiculosum, can be recombinantly expressed in E. coli cells. The enzyme is a glycoprotein composed of a single polypeptide chain with a molecular mass of about 37,000 Da. GFP is fused to the enzyme to measure expression levels. Brucato CL, Wong SS. Extracellular poly(3-hydroxybutyrate) depolymerase from Penicillium funiculosum: general characteristics and active site studies. Arch Biochem Biophys. 1991 Nov 1;290(2):497-502. doi: 10.1016/0003-9861(91)90572-z. PMID: 1929416.

Parts for Pseudomonas Putida

Type Part Link Name Functional Description
Basic BBa_25N4U2VJ RBS for Pseudomonas putida To improve the P. putida toolkit, Elmore et al., 2017 created a library of RBS and promoters for this strain. RBS JER07 showed protein production levels varied by ~6–7-fold from the highest to lowest strength RBS when optimized with various promoter variants. https://pmc.ncbi.nlm.nih.gov/articles/PMC5699527/#s0045
Basic BBa_251KQA88 Poly(3-hydroxyalkanoate) polymerase subunit PhaC with RBS for P. putida When expressed in conjunction with the genes PhaA and PhaB (from C.necator), this confers the ability to synthesize up to 13% (w/w) poly(3-hydroxybutyrate) (PHB) depending on the carbon source; all 4 genes are necessary for PHB production (https://www.uniprot.org/uniprotkb/P73390/entry). This coding sequence is combined with an RBS sequence (https://registry.igem.org/parts/bba-25n4u2vj) for translation within Pseudomonas putida - this provides an alternative to expressing in E. coli as described by previous iGEM teams (https://parts.igem.org/Part:BBa_K934001)
Basic BBa_2562DM8W Acetyl-CoA acetyltransferase - PhaA with RBS for P. putida Acetyl-CoA acetyltransferase - PhaA from Cupriavidus necator. Catalyzes the condensation of two acetyl-coA units to form acetoacetyl-CoA. Involved in the biosynthesis of polyhydroxybutyrate (PHB), accumulated as an intracellular energy reserve material when cells grow under nutrient limitation. https://www.uniprot.org/uniprotkb/P14611/entry. This coding sequence is combined with an RBS sequence (https://registry.igem.org/parts/bba-25n4u2vj) for translation within P. putida - this provides an alternative to expressing in E. coli as described by previous iGEM teams (https://parts.igem.org/Part:BBa_K934001)
Basic BBa_25I7O4MN Acetoacetyl-CoA reductase - PhaB with RBS for P. putida Catalyzes the chiral reduction of acetoacetyl-CoA to (R)-3-hydroxybutyryl-CoA. Source from Cupriavidus necator. Is involved in the biosynthesis of polyhydroxybutyrate (PHB), which is accumulated as an intracellular energy reserve material when cells grow under conditions of nutrient limitation https://www.uniprot.org/uniprotkb/P14697/entry This coding sequence is combined with an RBS sequence (https://registry.igem.org/parts/bba-25n4u2vj) for translation within Pseudomonas putida - this provides an alternative to expressing in E. coli as described by previous iGEM teams (https://parts.igem.org/Part:BBa_K934001)
Composite (Device) BBa_250E79FK PET to TPA in P. putida Inducible PETase LCC-ICCG (Leaf-branch compost cutinase), a highly thermostable PETase enzyme capable of high levels of Polyethylene terephthalate (PET) degradation (https://parts.igem.org/Part:BBa_K3478888). Designed to be used in P. putida under the Xyls-Pm expression system.
Composite (Device) BBa_25T2Z1R8 PHB production up-regulation with SerA in P. putida This part is designed to be used in Pseudomonas putida for the production of Poly-3-hydroxybutyrate, (PHB), a type of Polyhydroxyalkanoate (PHA), a biodegradable plastic which accumulates inside cells. PHB is produced via the biosynthesis of 3 enzymes encoded by the genes phaA, phaB and phaC. To up-regulate the biosynthesis pathway of PHB production, this part also contains Serine Repeat Antigen (SerA) gene (https://parts.igem.org/Part:BBa_K2086001). This gene codes for the protein 3-phosphoglycerate dehydrogenase, also known as PHGDH. The purpose of this gene is to increase the amount of NADPH available (Zhang et al., 2014) and thus upregulate the PHB biosynthesis pathway. We incorporated the Xysl/Pm (https://parts.igem.org/Part:BBa_K1973014) expression system which is recommended for P. putida. (Gawin et al., 2017)
Composite (Device) BBa_258G509X PHB production up-regulation with ZWF in P. putida This part is designed to be used in Pseudomonas putida for the production of Poly-3-hydroxybutyrate, (PHB), a type of Polyhydroxyalkanoate (PHA), a biodegradable plastic which accumulates inside cells. PHB is produced via the biosynthesis of 3 enzymes encoded by the genes phaA, phaB and phaC. To up-regulate the biosynthesis pathway of PHB production, this part also contains the ZWF gene (https://parts.igem.org/Part:BBa_K1674004). This gene codes for the protein Glucose-6-Phosphate-Dehydrogenase. The purpose of this gene is to increase the amount of NADPH available (Lim et al., 2002) and thus upregulate the PHB biosynthesis pathway. We incorporated the Xysl/Pm (https://parts.igem.org/Part:BBa_K1973014) expression system which is recommended for P. putida. (Gawin et al., 2017)

New Basic Parts

New Composite Parts

Biobrick name here


Diagram of the PET to TPA pathway in <i>E.coli</i>

We constructed a PET degradation system that enables E. coli to convert polyethylene terephthalate (PET) into terephthalic acid (TPA) and ethylene glycol (EG). This construct includes the part BBa_K3478888, encoding the engineered PETase that hydrolyzes PET, and BBa_E0040 which encodes for the GFP protein allowing for visual confirmation of enzyme activity. Part BBa_K3478888 is the LCC-ICCG gene with a H218Y mutation, which was introduced because it was shown to cause an increase in Petase activity (Orr et al., 2024). We confirmed the expression of this construct by measuring OD600 absorbance and calculated a maximum protein concentration of 3.79 ug/ml using a Bradford Assay.

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This construct in E.coli encodes a transporter system allowing TPA to enter E.coli and enzymes allowing for the breakdown of TPA into PCA. tpaK is the gene that encodes for the TPA transporter, allowing TPA to enter the E.coli cell. To ensure that the TPA transporter is on the cell before the production of TPA, two promoters were engineered into the construct: the T7 promoter (BBa_I719005) and a constitutive promoter (BBa_J23100). The T7 promoter is induced after the TPA transporters are synthesized. BBa_K808011 (tphA1) is the gene that codes for the enzyme terephthalate dioxygenase reductase. In this construct, BBa_K808012 (tphA2) and BBa_K808013 (tphA3) work together, along with tphA1, to form the complex terephthalic acid 1,2-dioxygenase system (TERDOS). TERDOS catalyzes the reaction that degrades TPA. BBa_K2013010 codes for the enzyme 1,2-dihydroxy-3,5-cyclohexadiene-1,4-dicarboxylate dehydrogenase (DCDDH) which decarboxylates the product of TERDOS to form PCA. This pathway of tphA1, tphA2, tphA3 and DCDDH are used due to its high efficiency in BL21 DE3 E.coli cell. The enzymes TPADO (tphA1, tphA2, tphB2) and DCDDH form a natural, sequential metabolic pathway in native TPA-degrading bacteria Comamonas sp. This feature allows those genes to have optimal heterologous expression, high catalytic efficiency, and proper protein assembly in E. Coli. Comparing with other constructs also derived from native TPA- degrading bacteria (such as Ideonella sakaiensis and Rhodococcus jostii RHA1), the TPA degrading genes derived from Comamonas sp showed the highest efficiency (Li et al., 2024).By comparing the area under the peak from HPLC for cells transformed with this construct with WT E.coli cells, we showed that the TPA transporter successfully took in TPA into the cell.

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This theoretical pathway in E.coli converts PCA into Acetyl-CoA and succinyl-CoA. The construct starts with the gene pcaK which encodes for the transporter allowing PCA to enter the E.coli cell. Then, the parts pcaGH, pcaB, pcaC, pcaD, pcaIJ, and pcaF code for various enzymes that are responsible for the degradation of PCA and producing the products Acetyl-CoA and succinyl-CoA. Due to time constraints, we were only able to design this construct and its performance is yet to be confirmed.

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This construct in E.coli converts Acetyl-CoA into PHB and then uses natural E.coli metabolic pathways to secrete the product out of the cell. The parts phaC, phaA, and phaB form the phaCAB operon which codes for several enzymes that convert acetyl- CoA to PHB. BBa_K2560091 codes for Phasin-HIyA, a protein combining phasin—which binds electrostatically to intracellular PHB—with the C-terminal secretion signal from the HlyA toxin. The HlyA tag hijacks E. coli's type one secretion system, causing the bound phasin-PHB complex to be transported out of the cell, allowing PHB to be excreted out of the E.coli cell and removing the need for cell lysis (Buchan et al., 2000).

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In P.putida KT2440 cells, this construct includes the same phaCAB operon used in the construct (biobrick name) in E.coli that converts TPA to PHB. We also included the part BBa_K1674004 that encodes for NADPH reductase which reduces NADP+ to NADPH. This increased concentration of NADPH makes PHB synthesis more efficient, since phaB encodes acetoacetyl-CoA reductase, which uses NADPH as an energy source. BBa_ K4728007 codes for phaF and has the same function as the Phasin- HIyA protein in the Acetyl-CoA to PHB pathway in E.coli, which is to secrete PHB out of the cell. The Phasin-HIyA used in that pathway was not used because the gene would not be translated into the final protein in Pseudomonas cells. Similarly, this removes the need for cell lysis and allows for the system to be reused.

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This construct in E.coli depolymerizes PHB into BHB. The construct includes an inducible promoter, RBS, coding sequence for PHAZ_TALFU, linker, coding sequence for GFP, and a terminator. Once we add IPTG, the system produces PHAZ_TALFU, which is a PHB depolymerase that catalyzes the hydrolysis of PHB into monomers of BHB, as well as GFP, which acts as a reporter. The linker will link the GFP molecule and BHB together, which can allow us to determine the levels of PHAZ_TALFU, and relatively the concentrations of BHB, by detecting GFP. By measuring fluorescence using FLUOstar Omega microplate reader we confirmed that this construct successfully expresses PHAZ_TALFU and synthesizes Polyhydroxybutyrate depolymerase. Using a fluorescein standard curve, it was also calculated that the highest concentration of Polyhydroxybutyrate depolymerase is shown to be 0.31 uM when 0.5 mM IPTG is induced for 16 hours.

References

Buchan, A., Collier, L. S., Neidle, E. L., & Moran, M. A. (2000). Key Aromatic-Ring-Cleaving Enzyme, Protocatechuate 3,4-Dioxygenase, in the Ecologically Important Marine Roseobacter Lineage. Applied and Environmental Microbiology, 66(11), 4662–4672.
Li, Y., Zhao, X.-M., Chen, S.-Q., Zhang, Z.-Y., Fu, Q.-S., Chen, S.-M., Chen, S., Wu, J., Xu, K.-W., Su, L.-Q., & Yan, Z.-F. (2024). Metabolic engineering of Escherichia coli for upcycling of polyethylene terephthalate waste to vanillin. Science of the Total Environment, 957, 177544.
MacArthur, D. (2017). The New Plastics Economy: Rethinking the Future of Plastics & Catalysing Action.
Namdev, P., Dar, H. Y., Srivastava, R. K., Mondal, R., & Anupam, R. (2019). Induction of T7 Promoter at Higher Temperatures May Be Counterproductive. Indian Journal of Clinical Biochemistry, 34(3), 357–360.
Orr, G., Niv, Y., Barakat, M., Boginya, A., Dessau, M., & Afriat-Jurnou, L. (2024). Streamlined screening of extracellularly expressed PETase libraries for improved polyethylene terephthalate degradation. Biotechnology Journal, 19(7), e2400021.
Stephens, E. B., Senadheera, C., Roa-Diaz, S., Peralta, S., Alexander, L., Silverman-Martin, W., Yukawa, M., Morris, J., Johnson, J. B., Newman, J. C., & Stubbs, B. J. (2024). A randomized open-label, observational study of the novel ketone ester, bis octanoyl (R)-1,3-butanediol, and its acute effect on β-hydroxybutyrate and glucose concentrations in healthy older adults. MedRxiv (Cold Spring Harbor Laboratory).
Joseph, T. M., Azat, S., Ahmadi, Z., Jazani, O. M., Esmaeili, A., Kianfar, E., Haponiuk, J., & Thomas, S. (2024). Polyethylene terephthalate (PET) recycling: A review. Case Studies in Chemical and Environmental Engineering, 9(100673), 100673–100673.