Results
The detection and quantification of microplastics is crucial for understanding their impact and developing effective mitigation strategies. To overcome this problem of detection and quantification of microplastics in water bodies, we devised an assay where an anchor peptide, which can easily attach to a plastic surface, along with luciferase helps with the detection of the microplastic by bioluminesce. The assay essentially consists of three parts. First, is the biotinylated anchor peptide which is attached to a streptavidin bead. Second, is the microplastic which gets captured by the anchor peptide and third, is the luciferase which is attached to the anchor peptide in our fusion protein. The oxidation of furimazine by our luciferase gives off a luminescent signal that can be detected with smartphone technology. Below we describe the results we obtained from the development of our assy.
Cloning and transformation Protein expression and purification Assay development References

Cloning and transformation

We went forward with the cloning of two insert constructs (Fig. 1), consisting of an anchor peptide fused with NanoLuc luciferase (NLuc) and another anchor peptide fused with eGFP. The eGFP construct was included as a control for the design of our NLuc construct. Our inspriration was taken from the eGFP construct developed by Ji et al. which has been conifirmed to bind polypropolene for microplastic detection. (Ji et al., 2021) The cloning of our inserts into the pET45b expression vector was initially met with challenges. In the early attempts, transformation of the gene of interest into Top10 E. coli cells resulted in either the absence of colonies or an excessive number of colonies in the negative control. These unexpected outcomes prompted us to critically evaluate and revise our cloning and transformation protocols.

Figure 1: The sequence inserts for our protein constructs visualised in snapgene. A) shows our NLuc construct of 828 bp. B) shows our eGFP construct of 1032 bp. In both you can see the details of the polypropolene binding anchor peptide and the linker sequence.

One of the key troubleshooting steps involved the analysis of restriction digestion. However, our initial efforts to verify successful restriction digestion using HindIII HF and SacI HF enzymes were unsuccessful. Despite conducting several gel electrophoresis experiments, we could not obtain conclusive evidence of successful restriction digestion. After weeks of careful consideration and brainstorming, we identified critical errors in the construct that had been ordered for this cloning project. These errors necessitated a revision of the construct, correcting the issues that had been identified.

With the revised construct in hand, we proceeded with the cloning process into the pET45b expression vector. This time, the cloning procedure was successful, resulting in the generation of colonies. To confirm the presence of the desired insert, colony PCR was performed, and subsequently, mini-prep was carried out on selected colonies. These purified plasmids were then sent for sequencing to validate the accuracy of the insert.

Upon receipt of the verified plasmid sequences, the constructs were transformed into Top10 E. coli cells. This set of transformed cells was employed for protein expression and subsequent purification, marking the successful completion of the cloning process.

Protein expression and purification

Once the sequencing results were validated, we started transformation in the BL21 cells which again gave good colonies as a result and then followed by pre-culturing and making glycerol stocks. This was followed by the main culturing and with an overnight IPTG induction for protein expression which was done at two different temperatures, 25°C and 37°C, to ensure and check which temperature is the best for cells to grow and express the protein. 25°C had the best results in terms of growth and expression. This was confirmed by doing cell lysis and performing an SDS page for the lysis product. The protein purification was done by batch purification method, as well as using ÄKTA columns to get more concentrated purified protein without contamination through other proteins (Fig. 2).

Figure 2: The SDSPAGE results obtained form batch purification for both the eGFP (A) and NLuc (B) fusion proteins. The gel images confirm the successful purification of our protein.

Assay development

The purified protein construct would then need to be thoroughly tested to ascertain binding specificity as well as functionality of the luciferase domain. This was done by analysing the luminescence of bound proteins on a 96-well plate with the help of a plate reader. A concentration gradient was tested alongside a blank containing no protein (Fig. 3).

Figure 3: The luminescence intensity was measured at different concentrations (µg/µl) pre-wash (A) and post-wash (B). Pre-wash luminescence values are significantly higher than that of the post wash, with an unusual decrease in luminescence intensity for the pre-wash samples at the two highest concentrations. Post-wash luminescent signal increases following a threshold protein concentration of 1µg/µl. This indicates the successful binding of our fusion protein. However, the optimal protein concentration is yet to be determined.

Pre-wash luminescence values are observed to be significantly higher than that of the post wash as a result of the high concentration of unbound protein. When analysing the post-wash samples independently of the pre-wash samples, it can be seen that binding does seem to occur, as the luminescent signal remains (though substantially weaker than that of the pre-wash). What can also be seen is that the binding capacity of the 96-well plate was not reached at a concentration of 8µg/µl, and significantly higher concentrations must be tested to obtain a standard curve based on our fusion protein.

An initial test was done to ascertain the binding of the protein construct to polystyrene. No luminescent signal was detected post-wash, supporting the specificity of our construct. However, due to time constraints, a full binding analysis with the appropriate controls, comparable to the one done with polypropolene plates, was not carried out. These results would enable us confirm our binding specificity.

Read more about our proof of concept here.

References

Bauten, W., Nöth, M., Kurkina, T., Contreras, F., Ji, Y., Desmet, C., ... & Schwaneberg, U. (2023). Plastibodies for multiplexed detection and sorting of microplastic particles in high-throughput. Science of The Total Environment, 860, 160450.

Ji, Y., Lu, Y., Puetz, H., & Schwaneberg, U. (2021). Anchor peptides promote degradation of mixed plastics for recycling. In Methods in Enzymology (Vol. 648, pp. 271-292). Academic Press.