To test whether cellulose binding domain fusion with peptide proteins can be synthesized efficiently, we tested several protein expression strains, namely E. coli KRX, HSM174 and Arctic Express (DE3).

We optimized protein synthesis by changing bacterial growing conditions. During all these iterations we learnt that our CBD-TA2 fusion protein has a tendency to go through degradation, when the synthesis is performed for a longer duration. We have also identified that there was no significant difference between strains, when growing CBD-TA2 at the most optimal conditions: 2 hours at 37 °C after induction with 0,5 mM IPTG. After choosing the most optimal recombinant protein synthesis conditions, we upscaled biomass production and prepared it for further protein purification steps.

We tested all of these strains by growing our bacteria for 2 or 4 hours at 37 °C or 16 hours at 16 °C after induction, optimized the inductor concentration, different growth mediums and lysis buffer pH and its compositions.

After reviewing our strategy and target protein sequence,we changed the DNA sequence for this peptide fusion protein. The sequence was cloned into a plasmid vector (pet29b(+)) and the synthesis of the protein was carried out under similar conditions as it was mentioned previously.

With the new sequence, we have successfully synthesised this protein by growing E. coli BL21(DE3) strain for 4 hours after induction with 0,5 mM IPTG. This protein has also shown an intensive fluorescence that was needed in other project steps.

After receiving the target protein sequence and successfully cloning it into a pet29b(+) plasmid expression vector, we tested the production of recombinant protein in different strains by using various inductor concentrations and different growing times.

After we saw that we are able to purify only a very limited amount of protein, we decided to upscale biomass production as well as to prolong the growth of the proteins and purify them again after testing if they are not showing any degradation signs.

During this engineering cycle we have learned that the problem might not be related to protein solubility, but the main issue here might be that these proteins tend to bind to plastic material and for this, we needed to use fully glass coated materials in order not to lose these proteins during the purification procedure. Although we were not able to do that, after first purifications, we stored target proteins in small glass bottles in order to prevent them from binding and thus decreasing obtained concentration.

Even if the amount of biomass was higher, the purification procedure became more complicated. The need to use larger purification tubes as well as prolonged attachment to Ni-NTA phase haven't shown any impact on protein concentration. Thus, we were not able to produce high amounts of our desired proteins.

For this, in this assay we decided to use small glass bottle columns instead of commercial tube columns (see them on Contributon).

Since we discovered that both of our domains have the ability to bind cellulose we decided to improve this assay, by using glass-coated microplates. In addition, we have also tested different CBD protein concentrations with the aim to determine the most suitable assay conditions.

We performed a cellulose binding activity test by using glass bottles with cellulose pieces inside and bacterial cell-free lysates. During this experiment we saw that our cellulose binding domain, attached to TA2 and LCI peptides, can successfully bind to cellulose-based paper.

For this assay, CBD-LCI CBD-TA2 composite parts were used on a glass coated microplate to identify saturation of CBD on whatman grade 1 chromatography paper.

Although we saw the fusion protein's ability to bind cellulose, our cellulose binding domain concentration was not sufficient enough to fully saturate cellulose paper. For this, we needed to measure, at what concentration cellulose membrane satures fully with this domain in order to find at what concentration protein of interest can fully saturate cellulose.

The concentrations of all purified proteins were assessed using a bicinchoninic acid (BCA) assay, and all standards and purified samples were tested three times to obtain greater accuracy. Protein purity was defined via SDS-PAGE gel image. Unmodified Whatman grade 1 chromatography paper was used as an immobilization platform for our CBD fusion proteins immobilization assays. After immobilization, absorbance at 562 nm was measured in order to evaluate protein binding efficiency onto the cellulose membranes. The constants showed that the protein of interest is able to bind to the cellulose with high efficiency, thus allowing us to move further with the experiments.

To achieve this, we purified larger amounts of protein thus obtaining higher concentration of the protein. Different concentrations were used in order to see, at what point our cellulose binding domain fully saturates cellulose.

Finding points of maximum protein saturation, in our case, is one of the key points on how we can utilize cellulose for these experiments. These experiments were an important part of creating our system as a whole and proving that it might work in a relevant environment.

We performed cellulose binding domain saturation experiments according to the 2nd iteration and found out that our cellulose can be fully saturated by CBDs. We tested 8 different protein concentrations, ranging from 100µg to 10µg of total purified protein and selected the most effective one. We successfully found out what is the lowest concentration that can reach a sufficient level of saturation.

To test whether fusion proteins can successfully bind to plastic, we performed an experiment with a microplate, made out of polypropylene (for LCI) and polystyrene (for TA2) with our old and newly synthesized GFP-plastic binding peptide constructs.

Due to low concentrations and/or low solubility of these fusion proteins, visualization of the reaction was difficult. In order to get the specific binding, further investigation is needed with purified proteins.

The experiment was carried out with cell-free extracts and specific polymer microplates. Fusion proteins were incubated in a microplate and after several washes, fluorescence was measured at excitation. 485 nm, emission. 520 nm in order to see specific binding. Tests showed that although fluorescence is slightly bigger in the wells, containing our peptides, the result is not repeatable and reliable.

The experiment was built with a new approach. Micro-sized plastic particles were incubated with bacterial cell free extracts. After the incubation and glass column centrifugation through a membrane, protein changes were visualised and evaluated via SDS-PAGE imaging.

During this experiment we learned that this invented protocol is a better solution to test peptide attachment using cell-free extracts. Microplate method requires purified proteins and glass bottle columns proved to be a better method to test interaction between plastic particles and peptides.

Glass bottles were incubated with plastic particles, cell-free extracts were used as a sample of choice because of the protein profile heterogeneity. SDS-PAGE analysis showed significant improvement from the microplate experiment. After that, a western blot technique was used with the aim to improve visualization of GFP fusion peptides.

The detection strategy will result in formation of sandwich-like complexes upon interactions between fusion peptides and plastic nanoparticles. The lower fusion peptides have an additional cellulose-binding domain which allows them to accommodate a high number of peptides on the testing surface. The upper fusion peptides are fluorescent-labelled, therefore they are able to provide an easy to interpret, light-based result.

We found unexpected results in our detection systems working principle. Questions were raised:

• 1) Is our detection system concentration dependent?
• 2) Do we need more comprehensive washing and incubation steps?
• 3) Is our nanoplastic sample good and soluble enough for our detection system to work efficiently?
• 4) How does the Fluorescence signal change when lower nanoplastic concentration samples are added?
• 5) Why does our control have a higher fluorescence than the lowest plastic dilution samples?

Testing of the detection system was done using glass coated polystyrene microplates. At the bottom of the well a round piece of Whatman paper (5.5 +- 0.5 mm) (Grade 1) was placed and CBD-peptide was immobilized on cellulose. Then, a buffer containing an aqueous colloidal dispersion of nanoplastics (nPs) was placed inside the well and incubated to bind to the immobilized proteins. After the liquid was removed, the purified GFP-peptide fusion were added to give fluorescent signals. During the experiment, we saw a couple unexpected samples, which include that our control sample had increased fluorescence, way higher than any of the nanoplastic particle dilutions. Besides that, fluorescence signals tend to decrease when nanoplastic concentration gets higher, instead of decreasing.

• 1) Proteins and experimental design stayed the same as in the first cycle.
• 2) Higher dilution samples were included in the test.
• 3) Incubation time was increased.
• 4) Washing steps were increased and fluorescence was measured every three wash steps.

What we found out during this experiment is that our system is sensitive and can successfully detect the lowest nanoparticle concentrations in a concentration dependent manner. When calculating the system's capacity, we found out that our system can detect ~0.6-1 µg/L and less of nanoplastic concentration, meaning this system is suitable to detect nanoplastic particles in an environment, where concentrations of these particles are lower than in laboratory settings.

The test flow stayed the same after the first iteration. When testing different washing steps we saw the change in a fluorescence that was unexpected. The fluorescence, after 3 separate trials, started to increase more at a certain point than a control sample. Interestingly, lower dilution nanoplastic samples (10x, 100x, 1000x, 10000x) showed a fluorescence lower than our control sample, but dilution of 500 000x (for PP) and 750 000x (for PS) showed a signal higher than the control and even lower dilutions started to gradually decrease from that peak. What we found was our system's capacity - a point where our system starts to detect specific concentrations of nanoplastic.

We built our bacterial detection system with protein EstA, antibody recognised sequence and our specific plastic binding peptides (namely parts BBa_K4380019, BBa_K4380020). The system was cloned into pet29b(+) bacterial vector. At first we needed to find out if our protein is expressed on our bacterial cell membrane.

This method was not accurate enough to show us whether our protein is expressed on bacterial membrane, because in SDS-PAGE imaging the protein of interest (~30 kDa) band was overlapping with the Proteinase K (~29 kDa), that was present in the sample. Although different SDS-PAGE gel concentrations were used, bands still overlapped to each other.

We performed whole cell proteinase K assay based on findings of Nicolay et al., 2012 [12] with and without bacterial cell lysis. The method that was described in literature, should have shown us two separate bands, based on our proteins of interest domains' size: an N-terminal passenger domain and a C- terminal anchor domain (~30 and ~35 kDa in size).

The experiment was performed using a FITC antibody, specifically designed for our system (NB600-525). The goal of this experiment was to evaluate if our proteins are expressed onto the membrane.

The first trials of proving our cell surface display system working principle showed unexpected results - our uninduced cells, after several attempts, produced higher fluorescence than induced cells.

We tested E. coli BL21(DE3) strain for our protein exposure onto the bacterial membrane. Several controls were used, including uninduced cells and untransformed BL21(DE3) strain cells. First, we tested the cell surface display system using a microplate reader to determine if our induced cells give out the highest fluorescence signal.

This time, the experiment included fluorescence microscopy in order to visualize our cells live in real time. This type of experiment was used to provide qualitative data over quantitative.

We learned, that fluorescence microscopy and fluorescence measurement in a microplate after incubation with antibody in all cases except for control cells proves that our cell surface display system works. Although uninduced cells emitted the most fluorescence out of all the cells, we believe this was because of nonspecific antibody binding. Thus, fluorescence due to antibody presence was evaluated as an unuseful application to evaluate bacterial presence in the sample.

For this experiment, several controls were used, including uninduced cells and untransformed BL21(DE3) strain cells. The cells were evaluated under fluorescence microscope and we saw that the cells, who were induced, were seen as the most bright. Uninduced cells, although producing a fluorescent visual, were not as bright as induced ones.

We built two reporter plasmids with fluorescent proteins (BBa_K4380010, BBa_K4380011 ) into the pACYC vector. This vector contained fluorescent protein and another one, pET based vector, contained cell surface protein. These two compatible plasmids were co-transformed and the fluorescence was tested.

We learned that this strategy suits our system better, because we could control it more easily and measurements are more accurate due to fluorescent protein presence.

We tested fluorescence of our newly co-transformed bacteria. Bacteria contained either mScarlet encoding plasmid or GFP encoding plasmid. Therefore, the evaluation could be successful between two different peptides. Firstly, the fluorescence of bacteria was evaluated by using a fluorescent microscope. After that, the first plasmid presence was confirmed by using antibodies, and the second plasmid presence was confirmed by changing fluorescent light and seeing mScarlet under different excitation and emission peaks.

We built a couple of epPCR primer sequences (BBa_K4380006, BBa_K4380007), which were used to amplify a specific peptide sequence using different epPCR techniques.

We learned that our epPCR was sufficient to produce a specific fragment with mutations. This fragment was used as a Megaprimer for the 2nd round of PCR.

We tested ep-PCR techniques, which utilized modified nucleotides and low fidelity of recombinant Taq polymerase. Different techniques were applied in order to get peptide libraries.

The first round of PCR generated a fragment with the desired mutations introduced. This amplified fragment—the megaprimer—is used in the second PCR along with the remaining external primer to amplify a longer region of the template DNA.

We learned that although our megaprimers had a good amount of mutation, the technique is not suitable for this protocol. During the second round of PCR on a plasmid vector, the vector itself gets mutated because, as we believe, there are not sufficient enough methods to purify a highly mutated megaprimer.

In the end, we have decided to skip the directed evolution after the first few iterations and prioritize other aspects of the project, as the other peptides already used in the system provided a satisfactory binding. In the future, to fully optimize the detection system for potential commercial or research applications we would suggest reiterating the directed evolution approach and possibly gain even more potent binders.

We tested adopting the megaprimer PCR technique for our peptides and we were able to successfully clone our megaprimers into the plasmid vectors, containing cell surface display proteins. Peptide libraries inside bacterial cells were isolated and sent for sequencing.

Bigger plastic particles needed first to be degraded by rough mechanic degradation. For this, a coffee grinder was used in a safe environment, in order to degrade plastic particles as much as possible. The particles were suspended in ethanol solution. After that, particles needed to be degraded more and for this a planetary ball mill needed to be used in order to gradually decrease plastic size. Filtration was performed with 5 µm PVDF filters with the aim to filter all of the particles, bigger than the defined size.

Signs of aggregation were seen in AFM micrographs, thus sonication is necessary each time before using samples.

Size of plastic nanoparticles was defined via atomic force microscopy (AFM). The microscopy showed that although we had successfully obtained nanoplastics, our samples were of high variability and included aggregated particles.

Buffer had to be made of low salt concentration, but needed to have appropriate pH so that it would not disturb other components of the detection tool. Linear alkylbenzene sulfonate (LAS) surfactant concentration was chosen to be below the critical micelle concentration to avoid micelle formation.

We decided that the concentration of plastics may be too high for all particles to disperse as a high amount of plastics were floating off the surface of water, furthermore, sonication was not enough to disperse particle aggregates.

We decided to find minimal surfactant concentration by adding little amounts. We mixed 0,05 g of nanoplastics with 100 ml of 5 mM Tris, 1 mM NaCl buffer and added 5 µl of LAS surfactant. Samples were sonicated to eliminate aggregation as much as possible.

To test this, we mixed 0,05 g of nanoplastics with 100 ml of 100 ml of 5 mM Tris, 1 mM NaCl buffer and added 2 µl of LAS surfactant.

The obtained results showed that our nanoplastic particles had a size variability and thus could not be measured correctly. Although measurements could not be performed with extremely high accuracy, we moved forward with our newly made plastic particles in order to test whether we can detect them.

To test whether we have successfully obtained nanoplastic particles of the correct size, dynamic light scattering (DLS) technique was performed. Samples were diluted for measurements and the hydrodynamic size of nanoplastics was measured by DLS, revealing that they were well dispersed in all cases.