The degradation of PET into terephthalate (TPA) and ethylene glycol (EG) occurs outside of the cell, and the degradation of TPA into PCA occurs inside the cell. Therefore, TPA needs to be transported into the cell through, what our project refers to as, a TPA transporter. However, since TPA is an hydrophobic aromatic acid, it does not diffuse over inact outer membrane of gram-negative cells (Nikaido 2003), such as E. coli. To increase TPA uptake, the cell membrane can be treated with chemicals such as n-butanol (Sadler & Wallace 2021), which has been used to increase cell membrane permeability for small substrates (Sadler et al. 2018) but has severe consequences on the cell envelope (Fletcher et al. 2016). Treatment with for example n-butanol, can lead to leakage of both periplasmic and cytosolic proteins, and therefore the use of a membrane transporter is thought to be beneficial for the single bacterium design our project focuses on.

Comamonas species strain E6 is a bacterial species that has the ability to take up TPA from its surroundings due to its TPA uptake system, consisting of the gene products from tpiA, tpiB and tphC (Hosakaet al. 2013). Hosaka et al. were the first to identify and characterize this TPA uptake system, and proposed that the system is a tripartite tricarboxylate transporter (TTT)-like system, a family of substrate-binding protein (SBP) dependent uptake systems. A prototypic TTT consists of a periplasmic substrate binding protein and two integral membrane proteins (Antoine et al. 2005). In the case of the TPA uptake system, TphC corresponds to the SBP (that binds strictly to TPA) and TpiA-TpiB to the integral membrane components (Hosaka et al. 2013, Gautom et al. 2021).

In the study by Hosaka et al. (2013), coexpression of the TPA transporter and the tph genes encoding TPADO and DCDDH in Pseudomonas putida Pp Y1100, showed that tpiBA (encoding for an operon containing both TpiA and TpiB) and tphC both were necessary for the resting cells' conversion of TPA. The TpiA-TpiB membrane components were also strongly suggested to interact with other molecules than TPA since disruption of the genes also caused growth deficiency of Comamonas sp. strain E6 on IPA, OPA, PCA and citrate. Additionally, TPA uptake seemed to be inhibited by the presence of PCA as well as complete inhibition by the protonophore CCCP. The latter observation made by the authors indicates that the system is dependent on proton motive force.

Plasmid pJCBAtG (Figure 1) was kindly provided by the authors behind Hosaka et al. 2013, at the Department of Bioengineering at Nagaoka University of Technology in Japan. pJCBAtG contains the genes for the TPA uptake system (tpiA, tpiB and tphC), TPADO complex (tphA1, tphA2, tphA3) and DCDDH (tphB) under P_m promoter control. The plasmid also contains tetracycline (Tet) resistance and was constructed for use in P. putida. The use of the P_m promoter control system in E. coli has been shown to in general generate much less transcripts than with the T7 promoter (Balzer et al. 2013). However, the P_m system has been shown to be capable of expressing high levels of proteins in high cell density cultivations (Sletta et al. 2004).

In this part of our project, the main focus was the TPA uptake system (TPA transporter) segments of the plasmid.

The obtained plasmid was resuspended in water and transformed into DH5α cells using the general transformation protocol, with 5 ng of the pJCBAtG plasmid. The transformation was successful, based on the growth on the Tet-agar plate (12 μg/mL Tet), which is shown in Figure 2.

Plasmid from a colony from plate B in Figure 2 was extracted (modification of protocol: 30 uL elution buffer and 5 min incubation time) and transformed into BL21 cells. The transformation was successful with growth of the cells with transformed plasmid (see Figure 3) and the cells were later used for protein expression.

Attempt for protein overexpression was performed to investigate if TPADO (see TPADO) and TPA transporter was overexpressed in E. coli BL21. No positive control was available for the P_m promoter system, therefore only a negative (uninduced BL21 with pJCBAtG) control was used.

The general protocol for protein overexpression was followed, with some modifications. The desired conditions for the overexpression were OD₆₀₀ 0.6, 30 ^oC and inducer 1mM m-toluate. These conditions and most of the modifications to the general protocol were based on the methods in Hosaka et al. 2013

Overnight cultures of BL21 transformed with pJCBAtG were diluted into two 1:100 solutions and two 1:10 solutions, and after approximately 3h, OD₆₀₀ measurements (see Table 1) indicated high enough values for induction.

Induction was done in 1:100 I and 1:10 I (hereinafter OD 0.8 I and OD 0.9 I) and no inducer was added to the (uninduced) controls (1:100 U and 1:10 U, hereinafter OD 0.8 U and OD 1 U respectively). All mixtures were incubated for 10h at the desired conditions, after which they were transferred to a fridge. 11h later, cells were harvested by centrifuging at 4 ^oC and 4000 rpm for 5 min, resuspended in 2 mL buffer A and lysis of the resuspended cells was done with the SDS lysis protocol. SDS-PAGE analysis was done with 7 μL of the four samples' supernatants, and stained by the quick version.

The SDS-PAGE analysis of the experiment showed no evidence for overexpression (see Figure 4) and the expression results were therefore inconclusive.

In an effort to try and overexpress the TPA transporter system within our E. coli chassis, we attempted to extract and ligate the tphCII and tpiBA genes into a pET24a(+) vector, which is more commonly used for overexpression in E. coli.

Referencing the plasmid map of pJCBAtG (see Figure 1) we were kindly provided, the tphCII and tpiBA genes were cut out using restriction enzymes NotI and XhoI, yielding a 4025 bp fragment. This fragment was then ligated into a NotI/XhoI linearized pET24a(+) vector of about 5302 bp with a flanking T7 promoter and terminator from the vector flanking the insertion sequence. The religation mixtures were subsequently transformed into chemically competent DH5α cells, plated onto LB-kanamycin plates and cultivated overnight. For analysis of cloning success, we chose eight colonies and conducted a digestion analysis, concluding that approximately half of them contained our target recombinant plasmid (see Figure 5).