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 :

• LacB.part1.FOR : “5’-agctccgtatgacacttgaaaaatttgtggatg-’3”
• LacB.part1.REV : “5’-gattctccttctgtgaagtcaatgatgatatcgt-’3”
• LacB.part2.FOR : “5’-acttcacagaaggagaatcgatcattttggc-’3”
• LacB.part2.REV : “5’-aagcttgtcgttatttatggggatcagttatatccatcg-’3”

Similarly for Fungal laccase :

• LacF.part1.FOR : “5’-CCAGGGCATGGGATTACAACGCTTCTCGT-’3”
• LacF.part1.REV : “5’-GCAAAGCCCACAGTTCCAAAGTTAGGATTCGC-’3”
• LacF.part2.FOR : “5’-AACTGTGGGCTTTGCAGGGGGAATAAAC-’3”
• LacF.part2.REV : “5’- agcttgtcgTTACTGATTGGCCTCTGATAAACCATCG-’3”

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).

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 :

• VectorB.FOR : “5’-ccataaataacgacaagcttgcgg-’3”
• VectorB.REV : “5’-agtgtcatacggagctcGAATTCG-’3”

Primers for the cloning of the fungal Laccase :

• VectorF.FOR : “5’-AATCAGTAAcgacaagcttgcggc-’3”
• VectorF.REV : “5’-AATCCCATGCCCTGGAAATACAAGTTTTCTCTG-’3”

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.

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 :

• VérifB.FOR : “5’-CATCGCCGCTTCCACTTTTT-’3”
• VérifB.REV : “5’-CCTCCGGATACCCGTCACTA-’3”

Primers to verify the presence of fungal Laccase :

• VérifF.FOR : “5’-ccacgcgggaaacg-’3”
• VérifF.REV : “5’-AGTGCAGCGGTAGGAG-’3”

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.

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).

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).

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.

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.

Peptides-GFP

To see our "Part collection", click here.

	Elution (PP)	Droplet (PP)	Elution (PE)	Droplet (PE)	Replica (PP)
PP1	X	X	X	X	X
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
PP3	X	X	X	X	X
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
PE3	X	X	X	X	X
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
PE6	X	X	X	X	X
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.

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.

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).

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.