Laccase part

In a first time, Bacterial laccase sequences have been received in two parts from IDT. Cloning will be done by Gibson.
Gibson strand assembly cloning is a cloning technique that does not rely on restriction sites as in traditional cloning techniques. Gibson strand assembly cloning relies on the presence of homologous regions at the ends of the DNA pieces.

In First, amplification of Bacterial Laccase part 1 and 2 have been done with two pair of primers :

Similarly for Fungal laccase :
For LacB part 1 we expect a size of 940 bp (Figure 1) and for LacB part 2 a size of 633 bp (Figure 2).

Figure 1 : Amplification of LacB part 1 and 2 on agarose gel 1%

Figure 2 : Amplification of LacF part 1 and 2 on agarose gel 1 %

Then, the plasmid pET-6His-TEV (containing his-tag), was linearized by PCR with primers homologous to the primers that allowed the amplification of the Bacterial or Fungal Laccase.
Primers for the cloning of the bacterial Laccase : Primers for the cloning of the fungal Laccase :
A 0.8% agarose gel was performed to verify that the plasmids were linearized and as we can see on Figure 3 that it is the case.

Figure 3 : PCR plasmid pET-6His-TEVlinearization for bacterial and fungal laccase clonings

Next, Gibson assembly was performed for bacterial and fungal Laccase and DH5alpha cells were transformed by the obtained products and plated on plates containing LB and 25 µg/mL ampicillin. Colonies have appeared and PCR on colonies were performed with a pair of primers, one of which hybridizes to the plasmid and the other to the cloned Laccase sequence.

Primers to verify the presence of bacterial Laccase : Primers to verify the presence of fungal Laccase :
For bacterial Laccase, on 16 colonies tested, 8 colonies were positives (Figure 4) because there were bands at 1039 bp which is the expected size. And for Fungal Laccase, on 8 colonies tested, 5 colonies were positives (Figure 5) because there were bands at 1140 bp which is the expected size.

Figure 4 : PCR on colonies to check the presence of bacterial Laccase

Figure 5 : PCR on colonies to check the presence of fungal Laccase

Then, plasmids containing the bacterial or fungal Laccase were purified and sent to sequencing which confirmed that there were no mutations on the sequences of bacterial or fungal Laccase. Next, BL21 cells were transformed with the plasmids containing bacterial or fungal Laccase in order to carry out production and subsequent activity tests. Production was made on 100 mL LB and 25 µg/mL ampicillin. Cells were broken by emulsiflex, centrifuged and purified on nickel columns. The expected size of bacterial laccase is 58.6 kDa and the size of fungal laccase is 56 kDa. An acrylamide gel stained with coomassie blue showed that the Laccases are in the insoluble Fraction (Figure 6).

Figure 6 : Acrylamide gel of the fractions after breakage (A.C), soluble (S) and non soluble (NS)

A further purification of Laccase was performed and the non-soluble fraction was solubilized with 0.5% SDS and further purified on the nickel column. Western blot were performed on bacterial and fungal laccase with antibody against his-tag. Only the bacterial laccase was purified (Figure 7), for the fungal laccase it did not work (Figure 8).

Figure 7 : Western blot purification of bacterial Laccase
A.B : after breakdown ; A.C : after centrifuged ; E : elution

Figure 8 : Western Blot purification of fungal Laccase

Concentration of purified bacterial laccase was calculated using the Beer-Lambert equation. The concentration is 0.4 µM. Then, the activity of the bacterial laccase was tested against ABTS only because it was too late to test on microplastics. As we can see on Figure 9, the purified bacterial laccase has no activity against ABTS since we obtained in 3 tests a curve similar to the control.

Figure 9 : Activity test of bacterial laccase

In parallel, we tried to design/improve the bacterial laccase so that it would be more active and that the active site would be more accessible but it was impossible to produce it.

The improvement of bacterial and fungal laccase was conducted by studying the tri-dimensional structure of wild type laccase from Bacillus Subtilis and Trametes Versicolor, then we came up with possible modification with one goal in mind : making the active site more accessible to microplastic so that the oxidation of microplastic take place more easily hence upgrading the degradation/oxidation activity of laccase. The models were after that predicted using AlphaFold and evaluated on chimera by calculating the distance between the oxidation site and microplastics and measuring RMSd of the global structure and specific amino acid.

The modeling resulted in 5 models (3 for the bacterial laccase, 2 for the fungal laccase) and one bacterial “upgraded” laccase was selected for production. Please find more about the different designs of laccase and the process behind the modeling in the following page : model.


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Elution (PP) Droplet (PP) Elution (PE) Droplet (PE) Replica (PP)
PP2 C1. 42 841 | C2. 10 544 C1. 15 599 | C2. 1034 C1. 5041 | C2. 7121 C1. 44 014 | C2. 1085 C1. 11 451 | C2. 3411
PP4 C1. 2908 | C2. 1868 C1. 15704 | C2. 1537 C1. 1843 | C2. 2057 C1. 29 975 | C2. 2935 X
PP5 C1. 42 631 | C2. 5206 C1. 9241 | C2. 722 C1. 12 680 | C2. 5483 C1. 8063 | C2. 1682 C1. 12 021 | C2. 4360
PP6 C1. 5310 | C2. 3064 C1. 21 356 | C2. 2285 C1. 8483 | C2. 43 811 C1. 16 727 | C2. 3558 X
PP7 C1. 12 721 | C2. 5995 C1. 1293 | C2. 433 C1. 18 055 | C2. 11 640 C1. 8119 | C2. 895 C1. 5319 | C2. 6080
SDS C1. 3441 X C1. 3902 X C1. 2000
GFP C1. 11 800 | C2. 4288 C1. 1130 | C2. 317 C1. 4428 | C2. 2640 C1. 27549 | C2. 523 C1. 6417 | C2. 2895
PE1 C1. 28 779 | C2. 9339 C1. 4751 | C2. 587 C1. 7603 | C2. 2979 C1. 14 235 | C2. 545 C1. 4453 | C2. 5225
PE2 C1. 6277 | C2. 5947 C1. 1172 | C2. 862 C1. 6461 | C2. 9479 C1. 11 200 | C2. 544 C1. 11 798 | C2. 5678
PE4 C1. 31 333 | C2. 9824 C1. 4884 | C2. 781 C1. 6556 | C2. 24168 C1. 1601 | C2. 693 C1. 9650 | C2. 5842
PE5 C1. 16 780 | C2. 17 292 C1. 25 173 | C2. 930 C1. 4600 | C2. 42 716 C1. 14 416 | C2. 889 C1. 5518 | C2. 2289
PE7 C1. 5779 | C2. 3740 C1. 12 602 | C2. 1845 C1. 5015 | C2. 3775 C1. 14 631 | C2. 1712 X

C1 : Concentrations at 350 ng/µL
C2 : 1/4 of C1

Phage display

As we said before, our objective of this part of the project was to find a molecule with a high affinity for polyethylene and polystyrene that is much bigger and much more specific to the type of plastic it adheres to than the peptides found in the litterature. For this, we decided to use the technique of phage display.
We used a phage library created by a French research team (the host-pathogen interaction team directed by Alain Roussel at the LISM in the CNRS of Marseille), and this library contained 5.1x10^6 different phages each exposing at its surface a different VHH. Four rounds of selection were carried out using the plastic of interest (polystyrene or polyethylene) as the target. The same experiment was performed in parallel but this time without the plastic of interest; this served as a negative control.

Then, the enrichment factor (Plaque-Forming Unit/mL) was calculated after each round of selection thanks to the enumeration of electro competent TG1 bacteria, which are not normally resistant to antibiotics but in this experiment are resistant to ampicillin. Indeed, when a phage infects a bacterium it transmits its genetic material (including the phagemid containing the VHH that it expresses on its surface and the gene conferring resistance to ampicillin).
In fact, during each round of selection, specific binders are selected out from the pool by washing away non-binders and selectively eluting binding phage clones. After three or four rounds, highly specific binding of phage clones through their surface VHH is characteristic for directed selection on immobilized antigen.
Ideally, after 3-4 rounds of selection there should be a 4 log between the enrichment factor of the negative control and between the antigen of interest. This indicates that the VHH seem to have a specific affinity for their antigen. In this experiment (Figure 1) we can see that after selection 2 the phages were enriched in both cases (for the plastic of interest) and for the negatif control. A 10 000 fold difference was observed for the control and a 1000 fold difference was observed for the plastic. In addition, after round 3 and 4 the enrichment factor did not increase any more in both cases. These results suggest that the VHH’s have a affinity for the surfaces in general.

Figure 1 : Enrichment factors calculated after each round of the phage display panning. Plastic (polystyrene or polyethylene) and PBS (control) were used as antigens during this protocol. These enrichment factors were calculated by counting the number of colonies of TG1 electrocompetent cells resistant to ampicillin and therefore carrying the phagemid containing a VHH. The enrichment factors are expressed in pfu/mL

We decided to continue the experiment even if the VHH’s exposed by the phages we enriched did not seem very specific. For dis we did a screening by ELISA. For this, we used a primary antibody anti-HA which will recognise the HA tag in the VHHs. We then used a secondary antibody anti-mouse-HRP which is conjugated to the Horseradish Peroxidase enzyme acting on various chromogenic substrates to produce many different colors. We used the BM Blue POD substrate and allowed us to detect a reaction by reading the optical density at 450 nm. This experiment was done with plastic as a target (polyethylene or polystyrene) and without the plastics (PBS) and IPTG - induction allowed the production by the TG1 cells of the VHH’s contained in the phagemids. We also did a negative and positive control of the ELISA test itself by not adding the VHHs into the reaction (negative control) and by testing an antigen already known for being a target of different VHHs in this library (positive control).

For each plastic (polystyrene and polyethylene) we tested 21 VHHs (Figure 2). We can see that for polystyrene we had several positive results compared to the positive and negative control of the ELISA but none of them seemed to have a very specific affinity for plastic because the PBS control was positive as well. We therefore choose 2 VHHs (in yellow) whose ELISA reaction was positive, therefore with an affinity for polystyrene but also for other surfaces. We named this 2 VHH PS-VHH1 and PS-VHH2 respectively.
For polyethylene, surprisingly, 2 VHHs seemed to have a specific affinity. In fact, we can see (in green) that the ELISA was positive for these VHHs when the target was polyethylene, and that the ELISA was negative when we used PBS. Therefore, we think that these VHHs are specific for polyethylene. However, it would be interesting to produce these VHH, purify them and test them. We didn’t have time to do so. We named these 2 VHH PE-VHH3 and PE-VHH4 respectively.

Figure 2 : Optical density read at 450 nm when performing an ELISA. This test was performed with a primary antibody Anti-HA (having an affinity for the HA tag linked to each VHH) and with a secondary antibody Anti-Mouse-HRP (reacting with BM Blue POD Substrate)

Then, after purification of the phagemid (phEN4) and sequencing of it, we were able to obtain the VHH sequence. Indeed, the VHH is inserted between the NcoI and BstEII restriction sites. Thus, after obtaining the protein sequences for each VHH (4) , we decided to align them (Figure 3). We can see that the sequences are organized into areas of highly conserved residues (highlighted in red), areas containing more or less conserved residues and hyper-variable areas (3). Indeed, this fits perfectly with the VHH structure. VHHs contain 3 hypervariable regions called CDR (Complementarity Determining Regions) responsible for antigen recognition and thus antibody diversity. These CDRs are spaced with scaffold regions, and are underlined in yellow (CDR1), green (CDR 2) and blue (CDR3).

Figure 3 : Structure based sequence alignment of PS-VHH1, PS-VHH2, PE-VHH3 and PE-VHH4. The CDRs are indicated by coloured bars

Then we wanted to look at the three-dimensional structure of each VHH. To do this, we predicted the structure using the Alpha Fold software. The Local Distance Difference Test (lDDT) is a superposition-free score that evaluates local distance differences of all atoms in a model, including validation of stereochemical plausibility. In all of our models (Figure 4), the lDDT score is superior to 70% for all of the residues except for the ones between 120-140.Therefore except for these residues, we can assume that our prediction is valid. In fact we can see that these residues that must be treated with caution form a long queue that doesn’t seem to fold. For the rest of the model we can see that the four VHH fold into a typical VHH structure. They are organized in antiparallel beta sheets. We can also see the loops that correspond to the 3 CDRs responsible for antigen recognition and thus antibody diversity.

Figure 4 : Structure of PS-VHH1, PS-VHH2, PE-VHH3 and PE-VHH4. The complementarity determining regions (CDR) are highlighted in yellow (CDR1), green (CDR2) and blue (CDR3).