We constructed a PET degradation system that enables E. coli to convert polyethylene terephthalate (PET) into terephthalic acid (TPA) and ethylene glycol (EG). Part BBa_E0040 is the GFP protein that allows for visual confirmation of circuit activity. Part BBa_K3478888 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. 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.

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. 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. Compared 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.

This theoretical pathway in E.coli converts PCA into Acetyl-CoA and succinyl-CoA. The gene PCAK codes for the PCA transporter on E.coli that allows PCA to enter the E.coli cell. Figure 4 shows the function of each PCA gene. The gene PCAGH codes for the enzyme protocatechuate 3,4-dioxygenase, which is responsible for the ring-cleavage of PCA, forming Beta- carboxyl-cis-cis-muconate. PCAB codes for the enzyme 3-carboxy-cis,cis-muconate cycloisomerase which catalyzes anti cycloisomerization, leading to the formation of γ-carboxymuconolactone. BBa_K2091002 (PCAC) codes for the enzyme γ-carboxymuconolactone decarboxylase which then, through decarboxylation, converts γ-carboxymuconolactone into Beta- ketoadipate enol-lactone. PCAD codes for the enzyme β-ketoadipate enol-lactone hydrolase which would catalyze a hydrolysis reaction that adds a water molecule on β-ketoadipate enol-lactone's lactone ring, forming β-ketoadipate. PCAIJ codes for the enzyme β-ketoadipate:succinyl-CoAtransferase, which forms Beta- ketoadipyl- CoA by adding a coenzyme A group on Beta- ketoadipate. PCAF codes for the enzyme β-ketoadipyl-CoA thiolase, which breaks the carbon-carbon bond in the middle of the β-ketoadipyl-CoA molecule to form succinyl- CoA and acetyl CoA. Acetyl-Coa would then go on to the next construct for further reactions. This pathway is efficient for acetyl-CoA production because they form a specialized, coordinated, and energetically favorable catabolic system for breaking down aromatic compounds directly into central metabolites. The enzymes in this pathway have high specificity for their substrates, minimizing side reactions and ensuring a smooth, efficient flow of carbon through the pathway (Buchan et al., 2000).

This construct in E.coli converts Acetyl-CoA into PHB then uses natural E.coli metabolic pathways to secrete the product out of the cell. phaC, phaA, and phaB come together to form the phaCAB operon. The phaCAB operon codes for an enzyme that converts acetyl-coa to PHB. Specifically, phaA codes for the enzyme Acetyl-CoA acetyltransferase that converts Acetyl-Coa into Acetoacetyl-Coa. phaB codes for the enzyme Acetoacetyl-Coa reductase which consumes NADPH as an energy source to convert Acetoacetyl-Coa into (R)-3-hydroxybutyryl-CoA. This is the reason why BBa_K1674004 is added. BBa_K1674004 is zwf gene which codes for the enzyme NADPH reductase that reduces NADP+ into NADPH. By adding the zwf gene into the construct, there will be a higher yield of NADPH which would result in a more efficient process of synthesizing PHB from Acetyl-Coa. phaC codes for the enzyme PHA synthase, which converts (R)-3-hydroxybutyryl-CoA into 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 which removes the need for cell lysis.

In P.putida KT2440 cells, we selected the Pm promoter which is coded by BBa_K4757009 (Kernel does not show the proper symbol for this promoter). phaC, phaA and phaB form the same phaCAB operon used in E.coli cells, which converts Acetyl-Coa to PHB. Since P.putida can naturally metabolize TPA and break down TPA into Acetyl-Coa naturally, only the PhaCAB operon is needed for the final conversion to PHB from acetyl-CoA.BBa_K1674004 encodes for NADPH reductase, which reduces NADP+ to NADPH, allowing for a higher concentration of NADPH in the system. 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. Phasin-HIyA is not used in this construct because the gene would not be translated into the final protein in Pseudomonas cells.

This construct in E.coli depolymerizes PHB into BHB. The system 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.

1. Testing for the construct of PET to TPA and EG (Synthesis of Petase)

Experimental Design

Construct Used:

Purpose

BBa_K3478888 is the LCC- ICCG gene, which codes for the enzyme PETase that can degrade PET into TPA and EG. To test this construct, the TPA concentration will be tested after the cell is lysed.

Procedure

The procedures for transforming plasmids that either encode the TPA transporter, TPA to PCA construct, PET to TPA, and EG construct are all identical. Our plasmids were all synthesized by GenScript. To transform the cell, we first prepared 250 µL of transformation solution. We then used a loop to transfer colonies of bacteria into the transformation solution. Then, we used another loop to transfer plasmid into the same solution and gently flick it to mix. The solution is then incubated on ice for 10 minutes, and after that transferred to a 42 degrees Celsius water bath for 50 seconds. After that, the tube is put back on ice for 2 minutes. Then, 250 µL of LB Broth was added into the tube, and 100 microliters of the solution was taken out from the tube and plated with a loop. The plate was incubated at 37 degrees Celsius overnight. 30 mL of LB + ampicillin and a colony of bacteria (from the plate using a loop) was then added into the falcon tube. The falcon tube is then incubated in a shaking incubator. The OD 600s are tested every 2 hours and when it reaches a maximum OD600, experiment Group Samples were induced with IPTG (0.1mM and 0.5mM) and a Control group of uninduced cells was also used for comparison. A Blank group containing Untransformed BL21 DE3 cells was also measured. After inducement, the samples are all centrifuged with 5000 rpm for 5 minutes. The supernatant is then taken out for Bradford Assay. The Bradford Assay Standard curve is generated under the Sangon Biotech’s Bradford Assay standard curve protocolhttps://store.sangon.com/productImage/DOC/C100530/C100530_EN_P.pdf.

Results

Calculations of Petase concentration under different time and IPTG concentration:

The PETase synthesized from our engineered E.coli cell has shown to be successfully synthesized and secreted out of the cell. The maximum OD600 level of 0.6 was reached after 240 minutes. From table 2 and figure 10, when the maximum OD600 of 0.6 is reached, the sample with 0.5 mM of IPTG induced has the highest absorbance of 0.19. This means the PETase concentration secreted out of the cell is highest when 0.5 mM of IPTG is induced. Bradford Assay was conducted according to the kit protocol described by Sangon Biotech. The standard curve generated and protein amount in ug/ml was determined as shown in figure 9. Using the standard curve generated from figure 10, the PETase concentration can be calculated in ug/ml. The highest PETase concentration is 3.79 ug/ml when 0.5 mM of IPTG is induced at maximum OD600 (0.6 at 240 minutes) shown in figure 9. There is also absorbance shown when the IPTG concentration is at 0.0 mM because T7 promoter is a fairly leaky promoter which means even without the use of an inducer, the T7 promoter still expresses the construct to a certain extent (Namdev et al., 2019).

SDS Page:

The measured protein concentration can be further verified as Petase using SDS- Page. The SDS Page is conducted with two samples Control (Not transformed) versus Transformed. The samples are centrifuged, and its supernatant is extracted out. TCA precipitation protocol for concentrating extracellular proteins is used on the supernatants. TCA precipation is required to concentrate the secreted proteins to a detectable level as these proteins are typically very diluted. TCA is used to get 10-50x more concentrated protein.

TCA Protocol:

Purpose:

This is the construct of TPA transporter in E.coli. BBa_K4728011 is the tpaK gene that codes for the TPA transporter. The TPA transporter allows TPA to enter the E.coli cell for further processes.

Calculations

The TPA transporter from our experiment result has been shown to successfully take in TPA into the cell. As shown in Figure 13 and Table 3, there is a decrease in the area under the peak as the time increases for the transformed sample. The area under the peak of HPLC is a measure of the TPA concentration in the extracellular space of each sample. A decrease in the area under the peak means a decrease in the TPA concentration in the extracellular space. This means the TPA entered the cell via the TPA transporter which resulted in a decrease in TPA concentration in extracellular space. As shown in Figure 13, the area under the peak for the control group (untransformed cell) is approximately constant over time which means the TPA concentration in the extracellular space is constant. This means the TPA did not enter the wild type BL21 DE3 E.coli cell. By using the standard curve shown in figure 12, the actual concentration of TPA in the extracellular space at each time can be calculated as shown in the calculation section. Furthermore, at the fifth time stamp (360 sec), the amount of TPA concentration of the experiment group increased significantly – it was decided to end the experiment and attribute the increase to cell lysis and toxicity of TPA: these cells did not have metabolic pathways to use TPA.

Calculations

4. Transformation of Chromoprotein Parts

We first designed parts that included each of the chromoprotein components as described by the 2011 Uppsala iGEM team's collection: http://2011.igem.org/Team:Uppsala-Sweden/2011 
Six different colors were chosen to form the palette for our agar art. These chromoproteins are expressed as proteins inside cells, typically used as reporters in synthetic biology, and are coded for by the following parts:

We placed the coding sequences for the chromoproteins under the control of the pBAD promoter and cloned them into a plasmid containing ampicillin resistance. We selected the pET3a plasmid to achieve strong expression of the chromoproteins, ensuring rich colors for our agar art.
Transformation was carried out using the heat shock method as described at:https://www.addgene.org/protocols/bacterial-transformation/ Cells were grown overnight on selection plates containing ampicillin.

Colonies that grew on the selection plates were inoculated into liquid LB broth containing ampicillin and grown for 12 hours before being transferred onto plates containing LB agar, ampicillin, and arabinose. Results are shown below:

Agar art could now be conducted using our transformed cells engineered to express chromoproteins in the presence of arabinose. Large square plates (9 cm × 9 cm) were prepared with LB agar containing arabinose and ampicillin to ensure chromoprotein expression. Some results are shown below:

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References