The discovery of MHETase started with the discovery of its host, Ideonella sakaiensis sp. nov.. Tanasupawat et al screened for bacteria in environmental samples that were able to utilize PET as a main source of carbon. From this, they managed to isolate the strain of Ideonella (Tanasupawat et al. 2016). Further sequence analysis was done on its genome, and an open reading frame of interest was found. What made this sequence so interesting is that it shared 51% amino acid sequence identity with a previously found hydrolase exhibiting PET-degrading activity. The protein was isolated and incubated together with PET film. After 18 hours, pits could be seen on the PET film, indicating that this enzyme could in fact break down PET. The product of this hydrolysis was identified as mono(2-hydroxyethyl) terephthalic acid (MHET), but it could only be found in minor amounts, indicating that it was rapidly metabolized. Further research led to the purification of another protein which seemed to be able to hydrolyze MHET with a high turnover rate. While several enzymes that can hydrolyze PET also have the ability to hydrolyze MHET, this specific protein did not have any activity when grown together with PET. In other words, it was only able to convert MHET into TPA and EG, leading to the conclusion that this was a MHET hydrolase, MHETase (EC 3.1.1.102) (Yoshida et al. 2016).

MHETase is believed to follow the reaction mechanism of α/β-hydrolases because of its fold. It will bind MHET and release TPA and EG.

While Ideonella sakaiensis is its native host, there have been reports of MHETase being successfully expressed in variations of E. coli (Janatunaim & Fibriani 2020, Yoshida et al. 2021). It has been investigated in terms of structure and reaction mechanism, providing a solid molecular background to its function.

PETase and MHETase have been relatively popular enzymes to work with in iGEM, so there have been several strategies in order to increase the efficiency of MHETase, usually together with PETase. For example, team HK_GTC tried the approach of linking PETase and MHETase together in 2021, forming a chimeric protein as they phrased it. They managed to successfully express and purify the protein, as well as perform an activity assay. However, the activity assay results were never compared to the WT proteins due to uncertainty of actual concentrations of chimeric protein that had been used (Team HK GTC 2021). Similarly, the DUT_China team designed a protein scaffold containing both PETase and MHETase as well as a fungal protein that alters the properties of PET surfaces in 2021. After successful ligation of these three proteins and expression, they managed to show that this protein scaffold was more efficient at metabolizing PET compared to using WT PETase alone (Team DUT China 2021). Another approach is to mutate the enzyme in order to increase its efficiency. This was done by team Exeter in 2019, but their enzymatic assays were never conducted (Team Exeter 2019).

The construct was designed in order to work in a standard BioBrick 3A assembly using a pET24a vector (Du et al. 2009). It contained a T7 promoter and terminator as well as a T7 extended RBS for expression in BL21 cells, together with a lacO so that protein overexpression could be induced. It also included a His-tag for purification purposes. An overview of the designed construct is presented in Figure 1.

Figure 1: Snapgene scheme of the MHETase construct.

Before cloning, the gene fragment was amplified in a PCR. Primers were designed to introduce extra base pairs in both ends of the construct, since the flanking regions of the BioBrick prefix and suffix would not be long enough to allow for efficient restriction otherwise. A limited gradient PCR and a gradient PCR were performed, where successful amplification could be seen in the gradient PCR (see Figure 2). PCR primers used were: Forward 5’-NNNNNGGAATTCGCGGCCGCTTCTAG-3’, reverse 5’-NNNNNNCTGCAGCGGCCGCTACTA-3’.

Figure 2: Agarose gel electrophoresis showing the results of the PCR for MHETase using the previously described primers.

The construct was then digested using restriction enzymes PstI and EcoRI from Thermo Fisher Scientific, according to their protocols. pET24 vector containing BioBricks was used as the backbone, producing a plasmid that could be transformed into NEB® 5-alpha competent E. coli cells and grown on kanamycin plates. It did take a lot of effort to create functional plasmids, with us having to repeat the procedure several times before finally being successful. Positive colonies were identified using colony PCR and the previously mentioned primers. Results can be seen in Figure 3. For future references, we recommend using highly competent cells since that seems to have had the biggest impact on our success. Plasmids were then purified and transformed into E. coli BL21 cells to allow for overexpression of MHETase.

Figure 3: Colony PCR was done on DH5α cells to identify colonies containing MHETase (2,2 kb). Successful colonies can be seen in row number 2 and 8.

In order to show that the plasmids from the two colonies did in fact contain MHETase, a gel analysis was performed. Plasmids were cut with PstI and EcoRI restriction enzymes, showing that the two colonies contain a DNA fragment of the same size as our MHETase construct. The results can be seen in Figure 4.

Figure 4: Agarose gel electrophoresis of plasmids purified from the two colonies identified during the colony PCR.

Lastly, a sequencing of our construct was made on three plasmids recovered from colonies stemming from colony 1 and colony 2. The sequencing result file can be found here.

Protein overexpression was induced using different concentrations of IPTG, at different OD₆₀₀, and at different temperatures in order to screen for the best options. IPTG concentrations of 0.01, 0.1 and 1 mM were combined with OD₆₀₀:s of 0.4, 0.6 and 0.8, together with temperatures of either 37°C or room temperature.

For positive controls, pET24a plasmids with IF3 were used. Cells were induced with either 0.1 mM or 1 mM IPTG at 37°C or room temperature. IF3 had, at this time, successfully been used as a reliable positive control in a previous experiment of ours. For negative controls, no IPTG was added to the IF3-producing cells.

After leaving the induction to run overnight, the cell cultures were centrifuged and the supernatant was removed. The pellet was resuspended in 50 µL of 1X SDS loading buffer and incubated at 95 °C for 10 minutes. Sample was centrifuged for 10 minutes. When trying to load the sample, it was extremely viscous and hard to load. 20 µL was taken out and diluted with 150 µL loading buffer, 10 µL was then loaded onto the gel.

The results for the negative and positive controls were as expected: No IPTG, no overexpression, while adding IPTG led to overexpression of E. coli IF3 (~20 kDa). However, the results show no overexpression of MHETase, whose band would be around 65 kDa. These results can be seen in Figure 5.

Figure 5: SDS-PAGE results showing overexpression.

If we had more time, there are several things that we would change in order to hopefully achieve our goals. The construct did not have the correct flanking regions surrounding the BioBrick prefix and suffix, leading us to perform PCR mutagenesis to fix this. As can be seen in the PCR product gel analysis above, PCR did not yield much product of correct size, leading us to have to repeat PCR and purify from gels in order to get enough pure DNA to work with.

Cloning was challenging, despite us using the very well-described method of BioBrick 3A assembly. We are unsure of what exactly could have caused this, but working with highly competent cells immediately would have saved us a lot of time.

Protein overexpression is where the most improvements could have been made. We based our variables on what we previously had known to work, and should instead have turned to articles describing successful overexpression and purification of MHETase, because it has been done several times successfully. One example is that Yoshida et al induced their cell cultures at 15°C (Yoshida et al. 2016, Yoshida et al. 2021). Similarly, Palm et al. let their cell cultures grow to an OD₆₀₀ of 1 when they induced, and at an OD600 2.5, they let the culture grow in 16°C overnight (Palm et al. 2019). Knott et al. kept their cells in 20°C after induction, which is similar to our room temperature (22 °C) (Knott et al. 2020). However, since experiments were done during the Swedish summer, room temperatures may have varied a lot. Either way, based on this previous work it would have been interesting to see if lowering the temperature that the cells were kept in after induction would have affected the protein expression.

A theory that did arise is that since we had trouble with our cloning and transformation, those successful colonies might have contained mutated sequences. If we assume that MHETase is more or less toxic to our cells, cells that contained the plasmid but did not express the functional protein would have had an advantage. In order to investigate this, we sequenced MHETase and its regulatory regions three times using two different sources. Due to the length of the MHETase sequence, we had partial coverage only. Those regions did not have any mutations we deem probable to affect protein expression nor function, but since we did not cover the entire length using the same source material, we cannot say for sure that mutations may not have played a part in the unsuccessful overexpression.