Notebook

Learn about our background research and the steps to achieve this project!

Main Constructs

PET to PHB

Figure 1

Figure 1: Research notes describing the paper on flotation technique to isolate PET.
Based on this source: (Guo et al., 2016).

    1. Problem with simple density separation
    • Plastics lighter than water (e.g., PE, PP) can be separated through density separation because they float
    • Other plastics (e.g., PET, PVC, PC, and PS) are all denser than water which means they will sink
      • Once sunk, they are indistinguishable from one another
    2. Solution modified flotation technique
    The principle of this method is to add/modify wetting agents in water to change the flotation behavior of plastics, allowing them to be separated. This process may need to be repeated 2 or 3 times to achieve full separation of a mixed plastic stream.
    • Chemical modification
      • Wetting agents can be used to treat plastic, making them more polar/hydrophilic to help with sinking
    • Specific protocol from paper
      • Use 3mol/L of NaOH to modify the plastic
      • Use 8mmol/L of DBS added to the water as a wetting agent (its function is to reduce the surface tension of the water)
    3. Outcome
    Using the specified method (NaOH modification and DBS wetting agent), PVC, PVC, PC, and PS plastics float while the desired plastic (PET) sinks.

PET to PHB

Reference Documents

    1. One-Step PET Degradation and PHB Biosynthesis Strategy
    • Rationale for co-cultivation
      • Using a yeast expression system alongside E. coli is advantageous because yeast does not require the addition of inducers, which reduces enzyme production costs (Liu et al., 2021).
    2. Key Enzyme for PET Degradation
    • The enzyme LCCICCG is highly effective for depolymerizing PET (Arnal et al., 2023).
      • Function - Converts PET into monomeric products Terephthalic Acid (TPA) and Ethylene Glycol (EG)
        • Our desired products are TPA and EG
      • Efficiency - Outperforms other enzymes
        • 98% conversion of PET into TPA and EG in 24 hours
    3. Optimized Reaction Conditions for LCCICCG
    • Optimization allows for a the required enzyme amount to be reduced by a third and a lower reaction temperature
      • Ideal reaction condition
        • 68°C, 0.1 M phosphate buffer at pH 8.0
      • Duration
        • At 68°, the reaction takes over 48 hours
    4. Protein production
    • Protocol for producing LCCICCG enzyme:
      • Host
        • E. coli BL21 (DE3) competent cells
      • Expression
        • Cultivation in ZYM auto-inducible medium for 23 hours at 21°C
      • Purification Process
        • Cell harvesting through centrifugation
        • Lysis in buffer (20 mM Tris−HCl, pH 8.0, 300 mM NaCl) through sonication
        • Clarification by centrifugation
        • Purification using TALON metal affinity resin
        • Washing with lysis buffer (+ 10 mM imidazole)
        • Elution with elution buffer (20 mM Tris−HCl, pH 8.0, 300 mM NaCl, 250 mM imidazole)
        • Buffer exchange to storage buffer (100 mM potassium phosphate, pH 8.0) using a desalting column
      • Analysis: Protein concentration determined by molar extinction coefficient; purity evaluated by SDS-PAGE.

Table 1

Table 1: Critical parameters to consider for an upscaling of the enzyme-based PET depolymerization reaction from the perspective of an industrial deployment(Arnal et al., 2023a).

PHB Production Optimization

    1. Overexpression of the zwf gene strategy
    • The zwf gene encodes for Glucose-6-phosphate Dehydrogenase (G6PDH)
    • Overexpression of zwf leads to increased levels of NADPH, which increases PHB production
    • This method provides a greater PHB production increase but also causes greater cell depression
    • The process must be aerobic (condition?)
      • Based on (Lim et al., 2003).
    2. Proposed systems for testing
      Comparison of 2 systems:
    • System 1: constant overexpression
      • Overexpression of zwf to increase NADPH and PHB production
    • System 2: switchable system
      • Overexpress zwf to boost PHB production
      • Includes the ability to switch off the overexpression to allow normal acetyl-CoA production to return to the regular TCA cycle to sustain energy by restoring normal metabolic flux when not producing PHB

    Source: (Shi et al., 1999)

    3. Metabolic engineering: Balancing NADPH and Acetyl-coa
    • Increases in the availability of both Acetyl-CoA and NADPH are necessary to achieve the maximum yield of PHB
    • Acetyl-CoA availability can be increased by…
      • Increasing fluxes from glucose to Acetyl-CoA
      • Inducing a blockage in the Acetyl-CoA-consuming pathway
    • NADPH availability can be increased by rerouting carbon fluxes to the Pentose Phosphate (PP) pathway (where zwf operates)

Figure 2: One of the major NADPH resources in E.coli is pentose phosphate pathway. Glucose-6-Phosphate-Dehydrogenase is the key enzyme to this pathway. Taken from: iGEM parts

    • Recombinant E. coli grows faster on gluconate than on glucose, indicating a high conversion rate to acetyl-CoA
      • Does not directly correlate to an increased PHB synthesis rate without sufficient NADPH
    4. Aerobic

Figure 3

Figure 3: E.coli genetic circuit with phbCAB and ZWF or GND
Figure 4
Figure 4: E.coli genetic circuit with PhnCAB Operon
Figure 5
Figure 5: Effect of zw and gnd gene amplifications on true cell mass (A) and PHB content (B) in the E. coli transformants. Cultivated in LB medium at the first stage for cell growth at 37°C for 24h, and then transferred to M9 medium containing 10 g/l of glucose and 5 g/l of NH,Cl for PHB accumulation at pH 7.0, 37°C, for 24 h at the second stage. Open circle, Transformant E. coli HB52 harboring the phbCAB operon only; closed triangle, transformant E. coli GHB; closed circle, transformant E. coli ZHB. Taken from: DOI

PHB Depolymerization

    1. Enhanced whole-cell biodegradation of PHB
    • Method
      • Co-expression of ORFCma with PHB Depolymerase (PhaZCma) in E. coli
    • Result
      • Co-expression induces efficient whole-cell biodegradation of polyesters
      • E. coli with only the depolymerase (pPHAZ) causes 80% weight loss in PHB films in 28 hours
      • E. coli with both the depolymerase and ORF (pORFPHAZ) achieves 80% weight loss in 21 hours
    • Enzyme Specific condition: Optimum pH for depolymerase PHAZ_TALFU is 6.0
      Based on: (Lee et al., 2018)
    2. Enzyme Screening and Evolution
      The objective was to screen and evolve PhaZ enzymes for higher activity and substrate specificity to degrade PHB into BHB.
    • Reference Papers
      • (Tan et al., 2012)
      • (Joho et al., 2024)
    3. Candidate polar molecules for petase system
      When selecting a polar protein candidate for a PETase system, factors considered included size, solubility, stability, and compatibility with the enzyme.
    • Human Serum Albumin
      • Most abundant protein in blood plasma- Globular protein
      • Weight is around 66.5 kDa
      • Water-soluble and biocompatible
      • Structure changes with pH
        • pH ~7.4: Maintains heart-like structure
        • pH ~3.5: Transitions into cigar-like shape
      • Its spherical structure may limit surface interactions
    • Type I Collagen
      • Most abundant protein in humans and animals
      • Weight is around ~3-6 kDa

Modifying E. coli to produce PHB from TPA

  • First Iteration

Figure 6

Figure 6: Original construct of TPA to PHB in P.Stutz.
In this construct, the promoter being used is the T7 promoter, BBa_B0034 as the RBS and BBa_K2560091 as the terminator. After researching Pseudomonas cells, we realized that the T7 promoter, BBa_B0034 and BBa_ K2560091 are not compatible with Pseudomonas cell.

  • Second Iteration

Figure 7

Figure 7: New construct with modified promoter
This discovery led us to find new promoters, terminators and RBS. We talked with Dr. deLorenzo where he recommended us to use xylS/Pm system where it offers tight regulation with minimal basal expression and high induction levels. After talking with Dr. deLorenzo, we also changed the organism we’re using from P.stuts to P.putida strain KT2440. P.stutz is harder to work with than P.putida since P.putida are more domesticated which is more optimal for genetic engineering than P.stutz where it has a more limited genetic tool box.

  • Third Iteration

Figure 7

Figure 8: Final construct modified to work in Pseudomonas cells
After sending the construct to the company Genscript for plasmid synthesis, we were told the original Phasin-HlyA (BBa_K2260002) would not work in Pseudomonas cells. The function of Phasin- HlyA is to tag and secrete PHB out of the cell. With this in mind, we found BBa_K4728007 which is PhaF. This has the same function as Phasin-HlyA, but it can work in Pseudomonas cells.

    The Processes

    • PET → TPA → PCA → PHB
    • TPA → PCA
    • PCA → PHB

    Based On:

  • (Li et al., 2024)
  • (Wells & Ragauskas, 2012)

Figure 9

Figure 9: PCA to PHB reaction pathway

  • Protocatechuate 3,4-dioxygenase (pcaGH):
      Sources:
    • (Part:BBa K2091001 - Parts.igem.org, 2016)
    • (UniProt, 2025)
  • 3-carboxy-cis,cis-muconate cycloisomerase (pcaB):
      Sources:
    • (EMBL-EBI, 2025)
  • 4-Carboxymuconolactone Decarboxylase (pcaC):
      Sources:
    • (Part:BBa K2091002 - Parts.igem.org, 2016)
  • ELH
      Sources:
    • (Gene - GCF_042159195.1, 2025)

    • FILLER/UNCOM. Change later pcaD (P. putida), catD (A. calcoaceticus)
    Beta-ketoadipate enol-lactone hydrolase
    TH: pcaF
    b-Ketoadipyl-CoA thiolase

Table 2: Table showing the enzymes involved in converting PCA into Acetyl- CoA

Experimental Design

PART 1: PET Linker

PART 2: Verify LCCICCG: PET to PHB and EG construct

  • PETase:
    • Optimization of reaction conditions for LCCICCG:
        Source: (Team:KEYSTONE/Engineering - 2020.Igem.org, 2020)
      • Combine 1 ml of a 0.69µM solution of purified LCCICCG protein (in 20 mM Tris-HCl, pH 8, 300 mM NaCl) with 100 mg PET powder and 49ml of 100 mM potassium phosphate buffer (pH 8) in a 100 ml flask
      • Incubate at 72˚C under 170 rpm agitation
  • PHB production:
    • Media: glucose;1 g, peptone;0.25 g, yeast extract;0.25 g, NaCl;0.01 g, KH2PO4;0.05 g, MgSO4;0.02 g (Krishnan et al., 2017, Mostafa et al., 2020).
  • PHB production (E COLI)
    • Optimum culture medium: 37.96 g deproteinated milk whey powder/l, 29.39 g corn steep liquor/l, and 23.76 g phosphates/l (r2 = 0.957)

PART 3: PHB to BHB (no PET)

  • Optimum reaction conditions:
    • pH = 6
  • Proposed conditions for PHAZ_TALFU optimization
    • Substrate: 0.1% PHB
    • Buffer: K₂HPO₄ (1.6 g/L)
    • Temperature: 27 ℃
    • Duration: Seven days
      • Based On: (Amir et al., 2022)

FBA Constraints

  • ZWF gene encodes for Glucose-6-Phosphate Dehydrogenase - overexpressing zwf produces increased levels of NADPH through the PP pathway, leading to increased PHB production
  • Try co-overexpression of sdaA - increase L-serine dehydratase 1 (L-SD1) enzyme
  • Try inactivating the phosphoglucose isomerase (encoded by pgi) gene
    • (Zhang et al., 2014)

Figure 11

Figure 11: Schematic representation of the SD and ED metabolic pathways in PHB accumulation recombinant E.coli. Dashed lines indicate multiple enzymatic steps. The bold lines indicate the enzymes of the SD pathway including reactions catalyzed by SerACB and SdaA. The enzymes that had been overexpressed in this work were shown in boldface. G6P, glucose-6-phosphate; FBP, fructose-1,6-bisphosphate; G3P, glycerahyde-3-phosphate; DHAP, dihydroxyacetone phosphate; 6PG, 6-phosphate-gluconate. Enzymes are as follows: Zwf, glucose 6-phosphate-1-dehydrogenase; Edd, phosphogluconate dehydratase; Eda, 2-keto-3-deoxygluconate 6-phosphate aldolase; Pgk, phosphoglycerate kinase; SerA, D-3-phosphoglycerate dehydrogenase; SerB, phosphoserine phosphatase; SerC, 3-phosphoserine aminotransferase; SdaA, L-serine deaminase I; PoxB, pyruvate oxidase; Pta, phosphate acetyltransferase; Ack, acetate kinase; PhaA, B-ketothiolase; PhaB, NADPH-dependent acetoacetyl-CoA reductase; PhaC, PHB synthase.

PET to TPA and EG (w/ LCCICCG/H218Y mutation)

Figure 12

Figure 12: This construct in E.coli converts PET to TPA and EG. Part BBa_E0040 encodes for the GFP protein that allows for visual confirmation of circuit activity. Part B LCC-ICCG/H218Y is the LCC-ICCG gene with a H218Y mutation. The introduction of a histidine to tyrosine substitution at position 218 in Petase shows an increase in Petase activity (Orr et al., 2024). LCC- ICCG codes for the protein Petase that degrades PET into TPA and EG. EG will not go on to further processes and will be discarded. Taken from: DOI

PHB to BHB (E.coli)

Figure 13

Figure 13: This is the construct of PHB to BHB Pathway

The system uses pTet to express the PHB biosynthesis enzymes (3 genes phaA, B, and C), followed by Phasin (binds to intracellular PHB and marks it for secretion outside of the cell). Also downstream of this promoter is LuxI for the production of AHL, which can activate pLux when cell density is high. Under the control of pLux is PHB depolymerase enzyme and TetR (repressor of pTet). This system will break down PHB into BHB as well as providing negative feedback to the first promoter to prevent over exhaustion of the cell and stopping PHB production.

PET to PHB (w/ zwf and LCCICCG/H218Y mutation)

Figure 14

Figure 14: This combines our constructs into one system for the breakdown of PET into PHB.

Experiment Logs

References

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