iGEM 2022 — Team aix-marseille

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# Team meeting summaries

| Date | Summary |
| --- | --- |
| 01.24.2022 | The team met up for the first time, some ideas and problematics were discussed. We have 3 main domains with different theme suggestions: Environmental (Toxic green algae, Brown marmorated stink bug, Neonicotinoids pesticides, Melting of the glaciers, Microplastic degradation); Therapeutic (Biofilms) and Medical diagnonsis (Cervical cancer, Endometriosis). |
| 01.31.2022 | The team went through the themes and discussed possible problematics that could be further analyzed. Concerning the medical diagnosis themes, suggested problematics revolved around finding better and non-invasive tests. In the environmental domain, the possibility to find harmless alternatives to nicotinoids and ways to eliminate sprayed pesticide were discussed. Moreover, in order to delay the melting of glaciers, the team discussed the ccreation of a gel capable of redirecting the sun's rays. Finally, bacteria-produced enzymes capable of degrading and digesting microplastics were considered. |
| 02.07.2022 | Team members continued to discuss and brought information and potential problematics for each theme. In the medical field, we suggested to find an easy and accessible test to be commercialized in developing countries. The goal is to have quick results to do more examinations if it’s necessary. In the environmental field, we would like to find a way to degrade nicotinoids that we can find in environment. In order to postpone the melting of glaciers, we would like to cover them with a film produced by a bacteria that we whiten in a second step. Concerning microplastic in Mediterranean sea, a problematic was given: how can we reduce durably their concentration ? |
| 02.22.2022 | More detailed presentations were made for the remaining topics. Different solutions were suggested. The team discussed the feasibility and will vote the next time in order to narrow down the possible topics. 4 topics remain and will be the object of further research and discussion. In the environmental domain: melting of the glaciers and microplastic degradation. In the health and diagnosis: uterus cancer and endometriosis. The advisors also presented the different roles and each team member was invited to pick a role. |
| 03.02.2022 | The team finally chose the theme: degradation of microplastics. In fact, this theme seems more fit as its a tangible problem that would interest the locals and would benefit our city. Thus, it would be easier and to raise awarness and collect funds. Moreover, every team member joined at least one of the 4 poles (business, events, social media and research). Advsiors mentionned an upcoming event in October (Fête de la science) for which we have to register. |
| 03.16.2022 | The advisors were asked to describe the different poles that are important for the good functioning of the team as well as their next missions on: communication, business and financing, events and human praticies, the Wetlab. After having described the objectives of each pole, each team member could choose on which point he wanted to invest himself during the project. The secretary of the team was appointed to keep all the minutes of the meetings. Another important point mentioned was the importance of everyone participating in the research in order to be as efficient as possible. Another objective of this meeting is to establish a problematic in order to orient the research. |
| 03.23.2022 | We have been thinking about setting up our first event "Le printemps des assos" to present our subject to students and maybe recruit new members. Everyone has exposed their ideas by thinking of communicating on the social networks of our presence in the next events. For the business pole, determination of the provisional budget. The research could progress, we decided to base ourselves only on 3 types of plastics, the most common (PP, PE, PS) and their means of degradation. |
| 03.30.2022 | This week, the business and events team sent several emails in order to look for funding and collaborations between different laboratories related to our project. The research must continue to focus on a concrete solution that the team will be able to implement during the Wetlab period. However the research of each one allowed the team to discuss the enzyme to be used for the degradation of the selected plastics. The research turns for the next week to determine how to adhere the enzyme to the plastic. This week we also made a schedule with the important dates of iGEM in order to be better organized and respect the deadlines. |
| 04.05.2022 | This week the events department has contacted and looked for potential partnerships or collaboration with different laboratories. We registered and shared our ideas to participate in a new event "La fête des sciences" to be held in October. Our idea is to use an artistic form to popularize the problem of ocean pollution by microplastics. We have also organized ourselves to participate in another festival on April 29th in Marseille in order to raise awareness of our problem and present our project. This could allow us to find financing thanks to the organizations which will be present. |
| 04.06.2022 | Our iGEM team has officially been created! We made a point on the progress of the research and made a point on the missing information and the questions which it is necessary to answer to advance in the project such as: what are the chemical reactions put in play? To think about the manipulations and the side effects... |
| 04.13.2022 | This week, the financing division continues to contact potential partners. The communication pole communicates on the events in which the team participated and on the recruitment of a new member to help during the Wetlab period. Finally, the events department is starting to think about the video that will present our project. For this week's research, we presented it in slide format to keep track and to be more organized. The data on the enzymes are becoming more precise, but the information on the chemical reactions is still missing. |
| 04.22.2022 | That week the team worked together to choose the shape and design of the video for our project, what we want to put in it and highlight. A meeting with the department producing the video is planned. A new event will take place in Marseille in June, the team is thinking about the activities they could propose during this event open to all public. |
| 05.04.2022 | Wetlab is starting soon!!! We have recruited the last person to participate in the lab experiments. This week the funding division has been dealing with private funding. We finally introduced the new iGEM Aix-Marseille 2022 team our social networks Facebook, Instagram and LinkedIn! The enzyme used has been determined: laccase. It lacks identified the type we would like to use and how to produce it. A plasticosme model will be used to attach the enzymes to plastic ! |
| 05.12.2022 | The laboratory experiments are starting soon, this meeting is to update the latest research elements, what to do and what are the last questions to be answered. First of all, to determine what type of laccase (molecule chosen) will be used to degrade the plastic. Then we need to determine exactly what enzyme to use and what it should do. Finally, how do you adhere the enzyme to the plastic? The members of the team divided the tasks in order to be more efficient. |
| 05.25.2022 | During this week we took stock of all the financial partners we have in order to evaluate a part of our budget. On the social networks our team posted awareness messages in the form of publications or interactive quizzes. A meeting is scheduled on May 30th between 2 researchers and the watlab team so that they can help them in their last questioning before the experiments. |
| 06.01.2022 | This week, the financing department has completed the necessary files for public financing. The orders for the experiments in the laboratory have also been completed. We communicated on social networks the subject chosen this year by the iGEM Aix-Marseille team and why we made this choice! All the team shared their ideas for the design of the new logo which will be designed by 2 students of the team. |
| 06.08.2022 | This week we had a meeting dedicated to Human Praticies in order to think about how to communicate about our topic with the population in order to best adapt our serious project to the feedback of surveys for example. The team decided on the name of the project "Seacare". The orders for the laboratory have been made. We concluded a collaboration with the "Delta Festival" in Marseille in order to participate to this event and to make us known. |
| 06.15.2022 | This week we have been working on the administrative procedures such as my scholarships and the safety form. The agreements for partnerships have been sent. We will participate to the bacteria book organized by the iGEM McGill team in Montreal. |
| 06.25.2022 | The file presented for public funding was defended by our president. This week, the communication pole has been organized to put online some posts to raise awareness about marine pollution with key figures, but also to present our subject more precisely. Other posts to present the events in which we participated in Marseille on June 13th. The team is also preparing a quiz in story to evaluate the knowledge of the people who follow us in relation to ocean pollution. |
| 07.02.2022 | This team member is training at the Gitlab so that we can start setting up what we will do for the wiki. We got some answers about the funding we had asked for. We made a return on the event in which we participated "the Delta Festival" between July 1st and 3rd. This allowed us to discuss with organizations about our project, to make us known and to create potentially new partnerships. We are preparing to come to a new event between July 7 and 9, organized by the iGEM team of Toulouse which proposed a gathering of the teams of France so that each one can present its project and discuss it. This week, the business pole has been working on its financing. We also decided who was going to be present at the event and set up a presentation of our project. |
| 07.09.2022 | The event in Toulouse went well, we are waiting for the feedback from the participants to communicate on social networks about it! The awareness quiz worked very well, many people participated. The experiments in the lab are still going on, the peptide cloning technique is working well ! |
| 07.16.2022 | This week we took stock of everything we have done for the iGEM project. We signed up for the "Fête des Sciences" event and thought about the activities we could do. |
| 07.20.2022 | This week we took care of the last financing. This year our university is organizing an event on the Mediterranean Sea with several speakers. We are organizing ourselves to know if we can participate. We made a quiz to propose collaborations to other iGEM teams. We have also finished the design of the poster explaining our project that we will be able to present at the next event like the "Fête des Sciences" on October 15th. We are starting to organize ourselves for the wiki by dividing the role of each one. |
| 09.21.2022 | A representative of each pole summarized the latest updates. Concerning the business pole, we were accorded the EU fund and we enumerated what is left to be paid. The lab team explained to the rest of the team the results. A poster using these results is to be made for 2 upcoming events on the 29th of september and the 3rd of october. The team went over the tasks to be done before the 30th. |
| 10.05.2022 | The team divided the last tasks to do before the wiki freeze. Everyone has a task to finish 3 days before the wiki -freeze. Moreover, the team agreed on a date to meet up for a picture and get the team tshirts and sweatshirts. Those who were at the Biotech Forum summarized how the event went. |

# Wetlab summaries

## Week 1 : 06.06.2022 to 06.10.2022

### Laccase

We did two cell transformations( JS228 in DH5α and JS321 in BL21) to produce a lot of plasmid. In addition to that, we did a test cell transformation (DH5α with chloramphenicol plasmid) to evaluate the bacterial competence. As we had a lot of colonies, we can say that our transformation did work well. Then we did a starter with the colony (BL12 to extract pET-GFP and DH5α to extract pET-6His-TEV). Meanwhile, we were writing some protocols and continuing research on enzymes with more accessible active sites. We did a miniprep of plasmid JS321 in BL21 strain and JS228 in DH5α and the first day (J+1) of the expression of GFP. Meanwhile, we continued the research on laccase and the ABTS to order, and we were trying to use chimera.

### Phage display

We also collected and studied different protocols for phage display used by different research teams all over the world in order to adapt them into the protocol our team will follow for affinity selection against plastic. In addition, our laboratory was located in a research center specializing in microbiology, so it was also an opportunity to talk to specialists in the field. We met Alain Roussel, head of the Host-Pathogen Interaction team, and Lycia Die Morini, team engineer and phage display specialist. We explained our project and our expectations and they offered to train us in the phage display technique and protocol.

### Laccase engineering

Interview with Vladimir Peylic
We discussed the possible use of pili type 4 and adhesin to make the cells on our project grip/join/adhere to micro plastic surfaces. Research on enzyme with more accessible active site. Selection of laccase from trametes versicolor and Bacillus subtilis as our proof of concept based on the following references :

* Fujisawa, M., Hirai, H. & Nishida, T. Degradation of Polyethylene and Nylon-66 by the Laccase-Mediator System. Journal of Polymers and the Environment 9, 103–108 (2001). [https://doi.org/10.1023/A:1020472426516](https://doi.org/10.1023/A%3A1020472426516)
* Srikanth, M., Sandeep, T.S.R.S., Sucharitha, K. et al. Biodegradation of plastic polymers by fungi: a brief review. Bioresour. Bioprocess. 9, 42 (2022). <https://doi.org/10.1186/s40643-022-00532-4>
* UTILIZING NATURAL COMPONENTS TO COMBAT ANTHROPOGENIC EFFECTS: BIODEGRADATION OF SINGLE-USE PLASTICS BY WHITE-ROT FUNGI by DENISE ANN HASSINGER A thesis submitted to the Graduate School—Camden Rutgers, the State University of New Jersey

## Week 2 : 06.13.2022 to 06.17.2022

### Laccase

Waiting for our genblocks corresponding to the Fungal and bacterial laccase.

### Phage display

We spent the whole week with the phage display specialists Lycia Die Morini and Flora Honoré ; they showed us the technique they normally use and together we were able to adapt their protocol so that we could use it to select the phages that have an affinity for plastic. We also ordered all the material necessary for this experiment, obviously following all their indications. The iGEM Check-in form was also completed this week, for the use of a bank of heavy chain only antibodies (vhh) derived from llamas.

### Laccase engineering

Went to an event organized to sensibilize children about plastics and its disastrous impact on the environnement. We held a booth offering 4 activities to raise awareness about plastics and their damage to the environment (specifically in the ocean):

* An improvised shark/fish operation table where children had to collect pieces of plastic, a fun activity during which children can learn/see that plastics end up in the environment, fragment and end up being eaten by fish.
* A pop quiz on recycling in France
* Bob the goldfish acted as a mystery box, the children had to remove plastic boxes from his stomach, identify them and analyze them.
* A timeline of the time of degradation of waste in nature

We had a goal in mind that was to be able to complete a engineering cycle in the 3 month we got at the lab : In order to do that we worked simultaneously on producing the wild type laccase and how we could potentially upgrade the laccase activity by making the active site/oxidation site more accessible to microplastics. We Searched for papers focusing on the specific laccase enzyme that we selected : Trametes Versicolor and Bacillus subtilis . The papers mentioned interesting information such as structural data, activity assays, mutation for specific amino acids and their consequences. We Started learning how to use chimera to visualize the structure of the bacterial and fungal laccases and alpha fold colab version 2 to be able to predict the structure of a modified laccase.

### Peptide

We did a PCR for oligo PP1 & PE2 (peptide + linker) to amplify them with pET GFP and obtain in the end the pET GFP with the oligo on it.
After that we made a test of 25 µL of the PCR to see if we have the strips that we were waiting for, as it worked, so we did a new PCR of 100 µL.
after that we did a test digestion of pET 6 His Tev with EcoR1 & HindIII, because we didn't know if the enzymes and the buffer that we have from last year were still working, after the migration on agarose gel and the verification using UV machine, we obtained 2 strips which means the digestion was done, then we did the same thing for the pET 6 His Tev for 200 µL and we did a DNA gel extraction and then we checked the OD and we got a small concentration of the vector because this technic wastes a lot of material, despite that fact, we continued our manipulations and we started a ligation of the vector pET 6 His Tev with the inserts digested followed by a transformation in DH5a to obtain clones.
We got 9 clones (4 PE & 5 PP), we did a PCR screen to see if get the insert in the plasmid (strips).
Production and purification of GFP only which will constitute a control for adherence test. Production in BL21 and purification of the GFP on nickel column. Then a migration on acrylamide gel was performed and allowed us to confirm our GFP purification.

## Week 3 : 06.20.2022 to 06.24.2022

### Laccase

Waiting for our genblocks corresponding to the Fungal and bacterial laccase.

### Phage display

During this week we started the first round of selection against polystyrene. We decided to use a phage library created by the phage display specialist that they have already used for their experiments. This library is called Loupio, and was created from the immunization of a llama with a protein X (which its name cannot be released) with a diversity of 5.1x10^6 pfu/mL.
However, we could not conclude from this first result because the bacteria containing the phagemids did not grow. This is most likely due to an experimental error as this was the first time this experiment was performed by us.

### Laccase engineering

We worked on upgrading the laccase activity by making the oxidation site more accessible for the microplastics based on the diverse information found on its structure. This was the beginning of our journey in modifying the laccase and predicting its structure using alpha fold, as it was our first time we had some difficulties at first and the predicted structures of the laccase were not great.

### Peptide

We did some starters of PE1 and PP1’s colonies (colonies 1, 2, 4 and 8). The next day we did the miniprep from the starter and checked the OD. We also did the transformation of colonies 1 and 8 into BL21. We did a digestion of the inserts and we add DpnI that we inactivate afterwards. We also did an other starter. We did continue with a miniprep, an insert cleanup and an other starter. We also did the migration of the vector pET 6 His Tev and extract the DNA on the gel. We did an other miniprep. We also did a PCR of the insert (100 µL final) and did migrate it on agarose gel. It was followed by an insert clean up, a digestion with DpnI. We also did a DNA cleanup that consisted of the purification of the vector in a mini colonne. We did a ligation of the vector with our peptide, followed by a killer digestion with SacI and finally a transformation in DH5α.

## Week 4 : 06.27.2022 to 07.01.2022

### Laccase

Reception of Bacterial laccase gblocks from IDT and start amplification of fragments for cloning in pET-6his, previously linearized, by Gibson Methods.
So the cloned Laccase has a Histidine-Tag if the cloning works.
Then, Transformation into DH5alpha.
The first results were not inconclusive because no clone grew after transformation.

### Phage display

This week we reconstituted the phage bank from the bacterial bank (TG1/pHEN4). In this library the bacteria have a pHEN4 phagemid which contains a VHH in its multiple cloning site. Each bacterium in this library has a different VHH. The phage library reconstituted and used for the rest of our experiments was a phage library of 1,02 x 10^12 pfu/mL.

### Laccase engineering

Pause/interruption of the design of a potential upgraded laccase to help in other experiments.

### Peptide

We prepare agarose gel 1% with TAE 1x. Then we did a migration on agarose gel of the colony of PP and PE. We did a PCR screen of PE and PP colonies. As our results weren't good, we did another PCR of our oligo PP and PE with the plasmid Pet GFP digested with HindIII and we did compare them with an oligo from team 2019 as a test to see if there is a problem with our oligo. We also did a digestion of pET GFP with HindIII because we seem to have 2 restriction sites of HindIII, we also did a digestion of PE in 4O µL and PP in 45 µL.

## Week 5 : 07.04.2022 to 07.08.2022

### Laccase

Try another strategy for cloning bacterial laccase by InFusion. For that, plasmids pET-6His were cut by restriction enzymes EcoRI and HindIII and for gene fragment by EcoRI/NdeI for one part and NdeI/HindIII for the other after amplification. Then, plasmids and fragments were incubated with InFusion mixture and transformed into DH5alpha.
Reception of Fungal Laccase gblocks from IDT and start amplification of fragments for cloning in pET-6his, previously linearized, by Gibson Methods and Transformation into DH5alpha.
We got colonies and made PCR on colonies to verify if colonies had the bacterial and Fungal Laccase but we got false positives.

### Phage display

We started the selection again against polyethylene, we did 2 rounds of selection but the controls were not good. It seemed like there was contamination by phages because we had ampicillin resistant colonies (the phage resistance) in our negative control of electrocompetent cells (TG1), that have no resistance at all . We also searched for the source of this contamination, we tested our culture media and our competent cells, and we disinfected our lab material.

### Laccase engineering

In order to engineer the optimized laccase we had to step back a little and learn more about the different tools that could be used in chimera : for example you can visualize the Hydrogens bond by following the next steps : select your structure, tools, structure analysis, FindHBonds. In order to simulate the presence of a microplastics you can follow the next step : tools, structure editing, build structure, select an atom, name it, give it coordinates if needed, then select the atoms, action, inspect and change the atom style from endcap to sphere and change its radius from 1.8 angstrom to the desired size (of a potential microplastic per example). You can also transform coordinates by following the next step : tools, movements , transform coordinate, or you could also select our structure then go to tools, movement, movement mouse mode and change it from normal to move selection.

### Peptide

We did a migration of the PCR that we made before to verify if they worked. As it was the case, we did an amplification of PP2 and PP3 and PE2 and PE3 at 50µL. We also did a sequencing analysis. (With the peptide with the right sequences, we did a ligation.) We did a cleanup of PE2 and PE3, PP2 and PP3, followed by a digestion and a ligation to the vector pET-6his-TEV. We then did a killer digestion and a transformation pET-6his-TEV with insert PE2 and PE3, PP1 PP2 and PP3 in the competent cell DH5𝛼. We also did a PCR (50µL) of PE4 and PE5 and PP4 and PP5. The inserts from the PCR are then cleaned up. Then it was followed by a digestion, a ligation to the vector pET-6his-TEV, a killer digestion and a transformation in DH5 𝛼. Then we did a cleanup of PE6 and PE7, followed by a digestion, a ligation to pET-6his-TEV. In parallel, we did a killer digestion of the mix from the ligation of PP2 and PE3 and a ligation of the insert PP1 to the vector pET-6his-TEV. We also did a PCR colony of PP1, PP2, PP3, PE2 and PE3, did a cleanup of PP6, PP7, PE6 and PE7 and then did a digestion. We also prepared 2 agarose gel and made a starter from the PCR colony (PP2C1, PP2C2, PP3C2, PP3C3, PE2C1, PE2C2 and PE3C5). We did a killer digestion of PP1. We also did a digestion of the vector pET-6his-TEV to make reserves. We did a transformation of pET-6his-TEV with insert PP1, PE5, PE6, PE7, PP6 and PP7 in the competent cell DH5𝛼. We did a miniprep with the starter of the PCR colony. We did a starter of PP4 clone 1, PP4C2, PP5C1, PP5C2, PE4C1 and PE4C4 and pET-GFP. We did a PCR colony of PP4, PP5 and PE4. We did PCR colonies of PP1, PE6, PE7, PP6 and PP7, followed by a migration on agarose gel. We did a miniprep of PP2C2,PP5C1, PP4C1, PE4C4, PP5C2, PE4C2; but as the concentrations were low, we did a re-concentration of the samples before sending them to sequencing.finally, we did an expression of PE1 clone6.

## Week 6 : 07.11.2022 to 07.13.2022

### Laccase

No progress cause of vacations and public holidays

### Phage display

We started again the first round of selection against polystyrene. This time all the controls were correct but the counting was difficult because we had a lot of colonies and our dilutions were not enough to allow us to count.

### Laccase engineering

Pause/interruption of the design of a potential upgraded laccase to help in other experiments.

### Peptide

We did a basic 6his Purification with pET expression of PE1C6.

## Week 7 : 07.18.2022 to 07.22.2022

### Laccase

This week, we I redo the cloning with InFusion for bacterial Laccase and Gibson for fungal Laccase and I try T4 Ligase for bacterial laccase too. I got no positive colonies for bacterial and Fungal Laccase with InFusion and Gibson but I got some positive colonies with T4 Ligase strategy after PCR on colonies. I sent it to sequencing after having recovered the plasmid by miniprep but the results showed that there was no bacterial laccase cloned.

### Phage display

We started again the first round of selection against polystyrene, this time doing more dilutions in order to be able to count the colonies. We discovered that even if we did more dilutions we were not able to count the colonies. We realized that it was an experimental problem, the dilutions were not done correctly. In addition, we adapted our protocol, because we also realized our protocol was not stringent enough. We decided to start our protocol by blocking both phages and plastic, and we also increased the numbers of washings before the elution.

### Laccase engineering

Design of 3 versions of the bacterial laccase, the prediction done in alpha fold was downloaded, then opened in chimera in order to analyze the structure of the modified laccase and set it side by side next to the wild type one, 3 measurement were conducted :

* The distance between the oxidation site and the microplastics
* The global RMSd of the aligned structure
* The RMSd of selected amino acid

### Peptide

We did a sequencing analysis of the peptides PP1, PP2, PP3, PP4, PP5, PP6 and PP7 to see if we have clones with the right sequence. We did a 50µL PCR of PE3, PP4, PP5, PE5 and PE7, then we did a digestion of the inserts and used Dpn1 to kill the enzyme. We did a ligation of the inserts PP5, PE5, PE7, PP4 and PE3 to the vector pET-6his-TEV, followed by a killer digestion and a transformation of pET-6his-TEV with insert PP5, PE5, PE7, PP4,PE3 in the competent cell DH5𝛼, It was also done with the miniprep of PP4 C1 and PP4 C2 (because the sequencing shows it was PP5). With the results of the transformation we did a PCR colony and a migration on agarose gel. It shows that we can use some clones to do a starter with them. Finally, in parallel, we did a transformation in BL21 of PP6C3, PP7C1, PE6C1, PP1C1 and PP2C2 and an expression of PE4C2 and PE2C1.

## Week 8 : 07.25.2022 to 07.29.2022

### Laccase

This week,we realized that the primers allowing the cloning of fungal laccase were false, so we ordered news primers verified previously with snapgene. I redo the cloning and I got, after PCR on colonies, 6 positive colonies on 8 and the results after sequencing shown that there were fungal laccase. So I began to produce with BL21.
For the bacterial Laccase, I continued to redo the InFusion and T4 clones but no success.

### Phage display

To gain time and because we felt our protocol was optimized at its best to do the selection against plastic we decided to start again the first round of selection but this time against polystyrene and polyethylene at the same time. The first round was successful, but there wasn’t a log difference between the enrichment factor of the selection done against plastic and the control. This was normal because it was the first round. We therefore decided to continue. But when a second round of selection was done, even if the enrichment factor did increase (for both polyethylene and polystyrene) it increased even more for the control. This is the opposite of what we expected. In fact, normally, the enrichment factor of the control should not increase.

### Laccase engineering

Selection of one of the designs of the bacterial laccase to test after exchanging with our team leader and order of the corresponding nucleotide sequence from IDT after optimization for E. Coli. Design of 2 versions of the fungal laccase, the prediction done in alpha fold was downloaded, then opened in chimera in order to analyze the structure of the modified laccase and set it side by side next to the wild type one, 3 measurement were conducted :

* The distance between the oxidation site and the microplastics
* The global RMSd of the aligned structure
* The RMSd of selected amino acid

### Peptide

We did a purification of PE4C2 and PE2C1. We also did a miniprep of PP4 C3, PP5 C1, PP5 C4, PE3 C2, PE5 C3, PE5 C4, PE7 C1, PE7 C2, PP4 C1 and PP4 C2 (what we have made with the starter) and send the samples to sequencing. We did a miniprep of PP2C2 and a test of competence of BL21. During this week, we also did some research on peptide test adhesion protocol. We also did a sequence analysis of the sample we sent to sequencing, the majority of them had a good sequence. We did a transformation of PE3 C2, PE5 C3, PE5 C4, PE7 C1, PP4 C3 and PP5 C4 in BL21, a dialyse of PE1C6, prepare some buffer (buffer 3 without imidazole and B-mercaptoethanol). Finally we did our first tests of adhesion protocol.

## Week 9 : 08.01.2022 to 08.05.2022

### Laccase

I tried for the first time, the production of fungal laccase. I have prepared a 100 mL culture of LB which, after growing to an OD of 0.7, has been induced with 1 mM of IPTG during 3 hours. Then, I made the different steps to purify and I made acrylamide gel.
Gel was not concluding because I had no laccase in my elution but no-induce and induce fraction showed that there was production of fungal laccase.
For the Bacterial Laccase, I redo Gibson methods and this time it works because I got a lot of colonies and after a PCR on 16 colonies, I got 9 positive colonies. Then, sequencing results showed that the Bacterial laccase was well cloned in pET-6His.
Then, I transformed into BL21.
Reception of Optimized Laccase gBlock with primers.

### Phage display

We re-started the second selection against polyethylene and polystyrene but the results were similar to the selection done the week before. We decided to continue because even if the number of phages in the control was increasing, the enrichment factor for the plastics was also increasing.

### Peptide

As the last transformation wasn’t conclusive, we did an other transformation of PE7 C1, PE3 C2, PP4 C3, PP5 C4, PE5 C3, PP6 C3, PP2 C2, PP7 C1, PP1 C1 and PE6 C1 in BL21. We also tried to find a website that allows us to calculate a molar extinction coefficient of sfGFP at 490 nm, which is the information needed for our adherence test. We prepared some acrylamide gel. We did a test of our protocol with random concentration just to try the manipulation in its all and to familiarize with the machines. We made a starter of PE3C2, PE5C3,PE7C1, PP2C2, PP4C3, PP5C4, PP6C3, PP7C1 and GFP. We continue our research on the adherence protocol to find the best way of doing it. We did a dialyze of the rest of PE1C6 & GFP. We did a transformation with those who weren’t successful (transformation of PP1 C1 and PE6 C1 in BL21) but this time by adding more plasmid and less LB. We also did a starter of PE7 C1, PE3 C2, PP4 C3, PP5 C4, PE5 C3, PP6 C3, PP2 C2, PP7 C1, PE1 C6 and GFP. (calculating some concentration of purified GFP and PE1 (for the adherence test). We made an expression for the starter of PE1C6, PE3C2, PE5C3,PE7C1, PP2C2, PP4C3, PP5C4, PP6C3, PP7C1 and GFP. It was followed by the purification of those proteins. We also prepare more buffers 1,2 and 3. We did a migration on polyacrylamide gel of PP4, PP5, PP6 and PP7 to see if we did a good purification of our protein.

## Week 10 : 08.08.2022 to 08.12.2022

### Laccase

This week, I made again a production of Fungal Laccase but this time after purification I made a Western-Blot. In the Western-blot, on the elutions fractions, I could see this time the Fungal Laccase but not at 55 kDa but at 25 kDa. It is possible that the laccase was degraded during the purification.
I tried for the first time, the production of Bacterial laccase. As the Fungal Laccase, I have prepared a 100 mL culture of LB which, after growing to an OD of 0.7, has been induced with 1 mM of IPTG during 3 hours. Then, I made the different steps to purify and I made acrylamide gel.
Here too, I could see production of Bacterial Laccase thanks to No-induced and induced fraction but nothing in elutions.
For the optimized Laccase, I started amplification of fragments for cloning in pET-6his, previously linearized, by Gibson Methods.
Transform into DH5alpha and I got colonies. So I made PCR on colonies and I got 13 positive colonies on 16.
Then, sequencing results showed that the Bacterial optimized laccase was well cloned in pET-6His.
Then, I transformed into BL21 and I Began to produce.

### Phage display

This week we continued our experience, we did selection 3 against polyethylene and polystyrene, but there was still a log of difference between the controls enrichment factors and the plastics enrichments factors (in favor of the controls). We therefore proceeded to do a fourth selection to see if we could improve our results, but this was not the case.

### Peptide

As we let our gel to colorate all week-end, we started the day with the discoloration of the gels of PP5, PP6 and PP7 and a recoloration and discoloration of PP4. But as the expected band was barely visible, we did a starter in case we’ll have to do the purification again. As the transformation of PP1C1 and PP6C1 in BL21 still wasn’t successful, we did once more. We also did a migration in polyacrylamide gel of PP2, PE3, PE5 and PE7, colored them and did a discoloration. We started the expression of PP4, then purified it. And we did a migration on polyacrylamide gel of PE1, PP4 and GFP, colored them and discolored them. We did a dialyse of GFP and PP3. (searching a way to calculate the concentration needed for the adhesion test). We did an adhesion test with PP3 and GFP.

## Week 11 : 08.15.2022 to 08.19.2022

### Phage display

This week we decided to re-do the selection 1 and 2 against polyethylene and polystyrene to compare the results to the panning done the weeks before. We concluded that the results were comparable (an increased enrichment factor for both the control and the plastics in each round of selection). We decided to stop at selection 2 and keep the results obtained during the weeks before (selection 1, 2, 3 and 4).

### Peptide

We prepared the calculus for the concentration we needed for the adhesion test. We did another transformation of PP1 C1 and PE6 C1 in BL21.

## Week 12 : 08.22.2022 to 08.26.2022

### Phage display

We stored 96 clones from the selections for each plastic at 96°C in order to conserve them. We also did a screening by ELISA of 21 of these 96 clones (chosen by hasard). For the positive clones, we purified their phagemid and sent it to sequencing in order to know the sequence of the VHH contained in it.

### Peptide

We prepared some buffer 3 without imidazole and 𝛽-mercaptoethanol for the dialyse. So we did a dialyse of PE1, PE2, PE3, PE4, PE5, PE7, PP2, PP4, PP5, PP6 and PP7. Meanwhile, we did some concentration calculus. We did an acrylamide gel migration of GFP, PE1, PE2, PE3, PE4, PE5, PE7, PP2, PP3, PP4, PP5, PP6 and PP7. And we continued our research on adherence tests to improve our protocol. We verified that the SDS2x we are going to use does not denature all of the protein or GFP, by checking with the magic box if we still have some fluorescence. We were thinking about measuring our adhesion with Tecan. For our new protocol, we did a pilot test with proteins with the highest concentration which are PE2, PP5, PP7 and GFP; we prepared a concentration at 350ng/µL and another one diluted at ¼. As the pilot test seems to work, we did the same thing with other peptides (PE1, PE4, PE5 and PP2).
We did a replicat of PE1, PE2, PE4, PE5, PP2, PP5, PP5 and GFP from the last test. We also did a specificity test of the adherence, which means to test peptide PE on plastic PP and peptide PP on plastic PE. We did an adhesion test with the proteins with the lowest concentration (PE7, PP4 and PP6) but with a concentration of 100µg/µL. we also did a triplicate of our protein (PE1, PE2, PE4, PE5, PP2, PP5, PP7, PE7, PP4, PP6 and GFP)

{% endblock %}

{% extends "layout.html" %}
{% block title %}Engineering Success{% endblock %}
{% block page\_content %}

During our project, we fully experienced the concept of the engineering cycle.
In order to make our design and the entire SeaCare project evolve, we tested each step of our thinking and analyzing our results was necessary to rethink our first idea.
Here, we will describe an example of how we worked with this concept. Indeed, it was especially applied during the search for a molecule adherent to the microplastics of interest.

# Peptide (engineering cycle 1)

**Design**
The idea of working with those peptides comes from the fact that they were used in some patents that shows their ability to adhere to PE and PP plastics. These peptides are all about 1,32 kDa. We decided to do the experiments with all of their peptides (7 PE and 7 PP).
To clone those peptides, we decided to link them with a linker which is also linked to GFP. For that, we use EcoRI and HindIII as restriction enzymes (**Figure 1**).

![](https://static.igem.wiki/teams/4189/wiki/engineering/gfp.png)

**Figure 1** : EcoRI and HindIII

**Production**
1- The 14 peptides used were found in patents where they are known for their supposed adherence to our 3 main plastics (polyethylene, polypropylene and polystyrene).
2- We used an E. coli strain (BL21) and a plasmid JS 321 (for the GFP).
3- An amplification of our inserts has been done by PCR.
4- Digestion and ligation: digestion by restriction enzymes EcoRI and HindIII were performed in order to access the plasmid and ligation was performed in order to connect the inserts to the plasmid.
5- A transformation in DH5 alpha was carried out in order to introduce the peptide genes, and thus transform the plasmid.
6- We verified our cloning results by doing a PCR screen.
7- A transformation in BL21 was done in order to be able to express proteins subsequently.
8- Then we had to purify our peptide after the induction; inoculation and centrifugation of E. coli.

**Testing**
To test them we imagined various protocols and contraptions but we settled on the following one. As the pilot test seems to work, we harmonized the concentrations at 350 ng/µL and prepared a 500µL solution for each peptide with a high concentration.
We then put 50 µL of the peptide solution on each plastic (polystyrene and polyethylene), testing both the peptide on its dedicated plastic but also on the other one, to verify to what extent it’s specific. Then, we used a Tecan : A Dedicated Fluorescence Reader; and used the SDS as a "white" when testing the plastic.
To analyze the results, we calculated from the OD504 we had at the beginning, every protein loss (on the washes, and the liquide we retrieved) by subtracting them. We replicated those steps to create a "replica" which verifies the quality and reproducibility of the protocol.

**Analysis**
All peptides adhere differently to plastic and their specificity of adherence also seems to be different. While all the PE peptides seem to specifically adhere to PE plastic, the results are the opposite for PP peptides which are not specific. Our experience shows that all the PE peptides that we were able to test all adhere to PE plastic. On the other hand, some PP plastics don't adhere to PP plastics while some do.

# Phage display (engineering cycle 2)

**Design**
Given the previous results our objective is to find a molecule with a high affinity for polyethylene, polypropylene and polystyrene that is much bigger and much more specific to the type of plastic it adheres. Therefore, we thought of VHH antibodies. A VHH antibody (or nanobody) is the antigen binding fragment of heavy chain only antibodies. They are very specific and their molecular weight is approximately 15KDa. We decided to do this experiment for polyethylene and polystyrene only because of the complexity of the experiment.

**Production**
1. The phage library was builded by a scientific research team by merging a VHH to a phage coat protein gene (here the pIII), causing the phage (here the M13) to "display" the VHH.
2. Binding: once exposed to the plastic, only a few phages interacted with targets in these libraries.
3. Washing: phages that are not bound can be washed away, leaving only those that have an affinity for the plastic.
4. Elution: the target-bound phage is recovered through elution.
5. Amplification: Eluted phages that exhibit specificity are used for direct bacterial infection and amplification of the recovered phage.
The cycle is then repeated two to three times to select the best binding sequence in steps.

**Testing**
For each plastic (polystyrene and polyethylene) we screened 21 VHHs 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).

**Analysis**
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.
In addition, their multiple alignment and their predicted three dimensional structure suggest that we did in fact produce 4 VHHs with 3 CDRs (Complementarity Determining Regions) responsible for antigen recognition and thus antibody diversity. However, it would be interesting to produce these VHH.

**Future experiments**
It would be interesting to produce these PE-VHH, purify them and test them to verify their specific affinity for plastic. If there are not, it would be interesting to redesign the phage library and the panning protocol.
If they are specific, it would be interesting to produce specific VHHs for polystyrene and polypropylene by adapting the protocol and the laboratory material.

{% endblock %}

{% extends "layout.html" %}
{% block title %}Experiments{% endblock %}
{% block page\_content %}

# Basic 6His purification with pET expression

* **Material**

+ E. Coli strain (BL21)
+ Plasmids JS 321 (GFP)

* **Day -1**

+ Transform BL21 strain with pET GFP on LB + Ampicillin at 37°C

* **Day 0**

+ Make a starter at the end of the afternoon = inoculate 10 mL of LB + ampicillin (4µL/mL) with one colony that you pick with a toothpick
+ Grow at 37°C overnight

* **Day +1**

+ Inoculate 100 mL of LB + 400 µL ampicillin with 1 mL of the preculture from the night in an erlen of 500 mL.
+ Incubate 1h ~ 37⁰C 180rpm → until OD= 0,6-0,8
+ Collect 1mL of non induced "NI" (before iPTG) in an eppendorf
+ Add 1mL of iPTG up to 1mM
+ Grow at 30°C during 3h with 180 rpm or until OD=3
+ Collect 1mL of induced "I" (after iPTG) in an eppendorf
+ If it’s too concentrated you dilute per 1/10 to check the DO
+ Divide into 2 falcons 50 mL
+ Centrifuge for 15 min at 4000 rpm
+ Throw away the supernatant and keep the pellet dry on the freezer

* **Day +2**

+ Resuspend pellet in 15 mL of buffer 1
+ Add DNAse (0,1 mg/mL) & lysozyme (0,1 mg/mL) only for emulsiiflex C5
+ French press/ emulsiflex C5 for cell lysis
+ Spin down at 45000 rpm for 30 min in the SS34 rotor
+ Wash 500 µL (1ml total -> (50% beads on ethanol) of Nickel beads in an Econocolumn Biorad
+ Retrieve 1ml of our solution “before N beads” and put it on the side
+ Close the bottom of the column and transfer the supernatant on it. (retrieve it on a falcon “non retenu”)
+ Incubate 15 min at 4°C on a wheel
+ Wash with 15 mL of buffer 1 (retrieve on falcon)
+ Wash with 5 mL of buffer 2 (retrieve on falcon)
+ Wash with 10 mL of buffer 1.(retrieve on falcon)
+ Elute 5 times with 0,5 mL ( ou 100 µL) of buffer 3 in eppendorf tubes (filled with 0,156mL of glycérol at 25%)
+ Homogenize, aliquote (200µL), freeze in liquid nitrogen and store at -80°C

* **Analysis**
Coomassie blue stained 12% SDS PAGE with :

+ 0,2 uDO600 of on induced and induced total cells
+ Equivalent 0,5 uDO600 extract (avant les billes de nickels)
+ Equivalent 0,3 uDO600 unbound
+ 5 µL wash 1,2 and 3
+ 2 µL of E1, E2, E3, E4 and E5

* **Buffers**

| Buffer 1 | Buffer 2 | Buffer 3 |
| --- | --- | --- |
| 20mM Tris-HCl pH 8.0 | 20mM Tris-HCl pH 8.0 | 20mM Tris-HCl pH 8.0 |
| 10mM imidazole | 10mM imidazole | 200mM imidazole |
| 200mM NaCl | 1M NaCl | 200mM NaCl |
| 2mM 𝝱-mercaptoethanol | 2mM 𝝱-mercaptoethanol | 2mM 𝝱-mercaptoethanol |
| H20 to 100mL | H20 to 50mL | H20 to 10mL |

Add 𝝱-mercaptoethanol just before use and keep buffers at 4°C when 𝝱-mercaptoethanol

# A highly stable laccase from Bacillus subtilis strain R5: gene cloning and characterization

Same protocol as "Basic 6His purification with pET expression" but Expression was induced by 4 mM IPTG in the presence of 10 mM copper sulfate. After 4 h of post-induction at 37 °C and 120 rpm, shaking was stopped in order to provide microaerobic conditions.

* **Material**

+ E. Coli strain (BL21)
+ Plasmids JS 321 (GFP)

* **Day -1**

+ Transform BL21 strain with pET GFP on LB + Ampicillin at 37°C

* **Day 0**

+ Make a starter at the end of the afternoon = inoculate 10 mL of LB + ampicillin (4µL/mL) with one colony that you pick with a toothpick
+ Grow at 37°C overnight

* **Day +1**

+ Inoculate 100 mL of LB + 400 µL ampicillin with 1 mL of the preculture from the night in an erlen of 500 mL.
+ Collect 1mL of non induced "NI" (before iPTG) in an eppendorf
+ Expression was induced by 4 mM IPTG in the presence of 10 mM copper sulfate. After 4 h of post-induction at 37 °C and 120 rpm, shaking was stopped in order to provide microaerobic conditions.
+ Collect 1mL of induced "I" (after iPTG) in an eppendorf
+ If it’s too concentrated you dilute per 1/10 to check the DO
+ Divide into 2 falcons 50 mL
+ Centrifuge for 15 min at 4000 rpm
+ Throw away the supernatant and keep the pellet dry on the freezer

* **Day +2**

+ Resuspend pellet in 15 mL of buffer 1
+ Add DNAse (0,1 mg/mL) & lysozyme (0,1 mg/mL) only for emulsiiflex C5
+ French press/ emulsiflex C5 for cell lysis
+ Spin down at 45000 rpm for 30 min in the SS34 rotor
+ Wash 500 µL (1ml total -> (50% beads on ethanol) of Nickel beads in an Econocolumn Biorad
+ Retrieve 1ml of our solution “before N beads” and put it on the side
+ Close the bottom of the column and transfer the supernatant on it. (retrieve it on a falcon “non retenu”)
+ Incubate 15 min at 4°C on a wheel
+ Wash with 15 mL of buffer 1 (retrieve on falcon)
+ Wash with 5 mL of buffer 2 (retrieve on falcon)
+ Wash with 10 mL of buffer 1.(retrieve on falcon)
+ Elute 5 times with 0,5 mL ( ou 100 µL) of buffer 3 in eppendorf tubes (filled with 0,156mL of glycérol at 25%)
+ Homogenize, aliquote (200µL), freeze in liquid nitrogen and store at -80°C

* **Analysis**
Coomassie blue stained 12% SDS PAGE with :

+ 0,2 uDO600 of on induced and induced total cells
+ Equivalent 0,5 uDO600 extract (avant les billes de nickels)
+ Equivalent 0,3 uDO600 unbound
+ 5 µL wash 1,2 and 3
+ 2 µL of E1, E2, E3, E4 and E5

* **Buffers**

| Buffer 1 | Buffer 2 | Buffer 3 |
| --- | --- | --- |
| 20mM Tris-HCl pH 8.0 | 20mM Tris-HCl pH 8.0 | 20mM Tris-HCl pH 8.0 |
| 10mM imidazole | 10mM imidazole | 200mM imidazole |
| 200mM NaCl | 1M NaCl | 200mM NaCl |
| 2mM 𝝱-mercaptoethanol | 2mM 𝝱-mercaptoethanol | 2mM 𝝱-mercaptoethanol |
| H20 to 100mL | H20 to 50mL | H20 to 10mL |

Add 𝝱-mercaptoethanol just before use and keep buffers at 4°C when 𝝱-mercaptoethanol

# DH5alpha/BL21 competent cells

* **Preparation**

+ Spread 100 µL on a LB petri dish and let grow overnight at 37°C
+ Pick one isolated colony and make an overnight starter of 5 mL of LB in a 50 mL Erlenmeyer
+ The next day, recover your starters and seed 1/100 = 0,05 mL = 50 µL bacterial cells in 200 mL of LB in a 1000mL erlen, then incubate 2-3h at 37°C and 180 rpm → until OD600=0,4-0,6
+ Recover your cells and pool them in cold 50 mL Falcon tubes
+ Prepare buffers 1 and 2 and put them on ice. Put Eppendorf tubes and pipette tips at -80°C for future use
+ Centrifuge tubes at 4000 rpm and 4°C for 10 minutes
+ Discard supernatant and resuspend each Falcon tube in 20 mL of buffer 1. Afterward, centrifuge at 3500 rpm and 4°C for 5 minutes
+ Discard supernatant and resuspend each Falcon tube in 8 mL of buffer 2
+ Take 100 μL aliquots in Eppendorf tubes and stock them at -80°C

* **Buffer 1**

| Product | For 40 mL |
| --- | --- |
| KAc (1 M) | 1.2 mL |
| MnCl2 (0.5 M) | 4 mL |
| KCl (1 M) | 4 mL |
| CaCl2 (0.1 M) | 4 mL |
| Glycerol (80 %) | 7.5 mL |
| H2O (sterile) | 19.3 mL |

* **Buffer 2**

| Product | For 8.5 mL |
| --- | --- |
| KCl (1 M) | 80 µL |
| MOPS (1 M) | 400 µl |
| CaCl2 (0.1 M) | 6 mL |
| Glycerol (80 %) | 1.5 mL |
| H2O (sterile) | 500 µL |

# Acrylamide Gel

* **Preparation**

+ Clean up the glass plates (1mm) with water only and wipe them down
+ Use ethanol to remove residues of acrylamide
+ Make the acrylamide assembly and verify first that the thin plate is smooth at the bottom to avoid gel leaks
+ Fill the space between the 2 plates with water to the end using a wash bottle and check after a moment if the water flowed a lot or not (if it flowed a little bit it’s okay your assembly is up to be used)
+ Remove the totality of water using a wipe
+ Meanwhile, prepare the reactifs of acrylamide gel
+ Follow the dosages of the first gel and fill it in the space between the 2 plates and leave a space of 1 cm
+ Fill the rest of space with water for few minutes and then remove the water like before
+ Follow the dosages of the second gel and fill the rest of space
+ Insert the brush on the top and let it dry a few minutes
+ Put the acrylamide assembly in a box completed with water and put it in the fridge

# LB Agar

* **Preparation**

+ Heat bottle in microwave at 300 W for 22 min
+ Let it cool down until you can touch the bottle with your hand
+ Prepare and work in a sterilized environnement
+ Poor approximately 25 mL per petri dish (until the surface is recovered and If there's bubble you can use a flame to pop them)

If you need an antibiotic, see to the table below :

| Antibiotic | color code | Volume to add in LB | Final [x] |
| --- | --- | --- | --- |
| Ampicilline 2 [x] :  - 25 mg/mL  - 100 mg/mL | 4 blues | 4 µL/mL | 100 µg/mL |
| Kanamycine 2 [x] :  - 10 mg/mL  - 100 mg/mL | 1 red | 5 µL/mL | 50 µg/mL |
| Tetracycline  15 mg/ mL | 1 black | 1 µL/mL | 15 µg/mL |
| Chloramphenicol  30 mg/mL | 2 green | 1.6 µL/mL | 50 µg/mL |

**Note** : if you’re using different antibiotics you should divide your volume according to what you need in separate erlenmeyer.

# Ligation

* **Preparation (20µL reaction)**
Adapt the volume according to what we have :

+ T4 DNA ligase Reaction Buffer (10X) : 2 µL
+ Vector DNA (4kb) : 50 ng
+ Insert DNA (Akb) : 37,5 ng
+ H20 to 20 µL
+ T4 DNA ligase 1µL
+ Work on ice
+ Add the mixture in a microcentrifuge tube
+ To mix the reaction, pipe it up and down and microfuge briefly
+ For cohesive (sticky ends), incubate at room temperature for 10 min. → our case (for blunt ends or single base overhangs, incubate at 16°C overnight or room temperature for 2h)
+ Heat inactivate at 65°C for 10 min
+ Chill on ice and transformation of the reaction into competent cells

# Mini prep

* **Preparation (NEB Monarch miniprep kit)**

+ Pellet 3 mL bacterial culture by centrifugation for 30s. Discard supernatant
+ Resuspend pellet in 200 𝞵L Plasmid Resuspension Buffer (B1) (pink)
+ Vortex ou pipet to ensure cells are completely resuspended. There should be no visible clumps
+ Lyse cells by adding 200 𝞵L Plasmid Lysis Buffer (B2) (blue/green). Invert the tube immediately and gently 5-6 times until color changes to dark pink and the solution is clear and viscous. **Do not vortex!**
+ Incubate for 5 min
+ Neutralize the lysat by adding 400 𝞵L of Plasmid Neutralization Buffer (B3) (yellow). Gently invert tube until the color is uniformly yellow and a precipitate form. **Do not vortex!**
+ Incubate for 2 minutes
+ Clarify the lysate by spinning for 10 minutes at 13000 ppm
+ Carefully transfer supernatant to the spin column and centrifuge for 1 min. Discard flow-through
+ Re-insert column in the collection tube and add 200 𝞵L of Plasmid Wash Buffer 1. Plasmid Wash Buffer 1 removes RNA, protein and endotoxin. Centrifugate for 1 min. Discard flow-through
+ Add 400 𝞵L of Plasmid Wash Buffer 2 and centrifugate for 1 min
+ Centrifugate for 1 min one more time
+ Transfer column to a clean 1,5 mL microfuge tube. Use care to ensure that the tip of the column has not come into contact with the flow-through. If there is any doubt, re-spin the column for 1 min before inserting it into the clean microfuge tube
+ Add 30 𝞵L Hot Water (deionised) (70°C) to the center of the matrix. Wait for 1 min, then spin for 1 min to elute DNA

# PCR on colonies

* **Context**
Bacteria were transformed via a vector to produce specific peptides to the adhesion to 2 types of microplastics (PP and PE).The empty vector or the one with the insert confers an ampicillin resistance to the bacteria that have integrated it, we want to know if the inserted plasmid is empty or has the insert for the 9 colonies present on the culture box. Oligo T7 and T7 reverse present on the vector upstream and downstream of the multiple cloning site, allow to make a colony PCR and to amplify. If the vector is empty, amplification by T7 primers gives fragments too small to be visualized by electrophoresis (migration on agarose gel: minimum value observed: 200pb). If the vector has only the T7, we get a fragment of about 40pb (not observable on the gel), with the insert it will be a fragment of 800pb.

* **For each colony**

+ Prick a colony with a toothpick then screen it to make sure we don’t lose any (use a grid pattern with numbers adapted to the petri dishes)
+ Work in a sterile environment
+ Prepare 11 tubes: 9 colonies + 2 controls (+ and -)
+ 1 PCR tube for verification = 13 µL
+ Do the preparation mix for 12 tubes to make sure we have enough
+ 12\*13 = 156 µL of mixture to be prepared (150 µL to make the calculation easier)
+ The master mix is 2X, it needs to be diluted 2 times (because we have a mixture for 50 µL final)
+ Put the mixture down below in each PCR tube :

|  |  |
| --- | --- |
| Master mix | 75 µL |
| Oligo T7 | 3 µL |
| Oligo T7 reverse | 3 µL |
| Colony | 1 |
| H20 (150 µL) | 75 µL |

+ Start the PCR program with Tm = 55°C, elongation 1 min/kb

# PCR

* **Preparation**

+ Mix components:

  |  | for 25 µL | for 50 µL |
  | --- | --- | --- |
  | 10 µL Forward Primer | 1,25 µL | 2,5 µL |
  | 10 µL Reverse Primer | 1,25 µL | 2,5 µL |
  | Template DNA | Variable\* | Variable |
  | Master mix | 12,5 µL | 25 µL |
  | H20 | to 25µL | to 50µL |

  \*We did use 0,5 µL on 13-06-2022

+ Assemble all reaction components on ice
+ Gently mix the reaction and collect the liquid at the bottom of the tube with a quick spin
+ Transfer reaction quickly to a preheated thermocycler (98°C)

+ Initial denaturation: 98°C, 30s
+ 25-35 cycles:

- 98°C, 5-10 s
- 50-70°C, 10-30s
- 72°C, 20-30s/kb

+ Final extension: 72°C, 2 min
+ Hold: 4-10°C

# Adherence

* **Preparation**

+ Cut pieces of plastic: 9 x 9 mm (for PP, PE, PS)
+ Put it on a glass plate (becher)
+ Check the OD504 of the liquid containing the proteins
+ Add 1ml of liquid on the glass plates (A and B) (ex: 100μL E2 + 900 Buffer 3, et 400μL E2 + 600μL Buffer 3)
+ Put them on a rocking stirrer
+ Plate A → After 30 mins (short term analysis) , retrieve the liquide and check the OD504 → place the tape in a fluorometry machine tank and add 1 mL of deionized water (the same used to make the «white» on the machine), and check the OD504 once more
+ Plate B → After 3 hours (long term analysis) , retrieve the liquide and check the OD504 → place the tape in a fluorometry machine tank and add 1 mL of deionized water (the same used to make the «white» on the machine), and check the OD504 once more
+ Repeat those steps for every peptides and plastics you have
+ To analyse, calculate from the OD504 you had at the beginning, every protein loss (on the washes, and the liquide you retrieved) by substracted them
+ Use your plastic and deionized water on your tank to act as a «white» when testing the plastic
+ **Use, a solution with different concentration, to know to what extend it’ll help the adherence**
+ To do so, check the OD600 on a sectrophotometer (→ it will be too high for the fluoro)
+ To modify the concentration, use the buffer you previously used during the purif.
**Note** : Verify the concentration of salt (more or less) for plastic binding+ Do the same, with only GFP on the plastic (to know if the GFP has an adherence property)

# Transformation

* **Preparation**

+ Thaw the cell tubes on ice (cells/ bacterias = 100 µL)
+ Recover the needed number of competent cell tubes, adding an additional negative control tube
+ Add DNA (1 µL) into the competent cell tubes
+ Incubate tubes on ice for 20 min
+ Incubate tubes at 42°C for 1 minute for thermal choc
+ Leave tubes on ice for 5 min
+ Add 1 mL of LB per tube, then incubate for 1 hour at 37°C and mix (850 rpm)
+ Centrifuge tubes 4 min and 4000 rpm at room temperature. And get rid of 900 µL of supernatant
+ Resuspend and spread cells (100 µL) on Petri dishes with the desired antibiotic and incubate overnight at 37°C

|  |  |  |
| --- | --- | --- |
| Transformation | 20 µL DH5α + 1 µL JS228 | Ampicillin |
| Transformation | 20 µL BL21 + 1 µL JS321 | Ampicillin |
| Test | 50 µL DH5α + 3 µL plasmid (100 pg/ µL) | Chloramphenicol |
| Transformation | 1 tube of DH5α + 1 µL JS228 | Ampicillin |
| Transformation | 1 tube BL21 + 1 µL JS321 | Ampicillin |
| Test | 1 tube DH5α + 3 µL plasmid (100 pg/ µL) | Chloramphenicol |

* **Note**

+ Spread with balls when you have a little quantity of DNA = picogramme, or when you want to do a cloning to have isolated colonies
+ Spreading using streak spreading when you transform a plasmid in cells to avoid a bacterial mat

# Laccase activities

Laccase activity was assayed using 2,2'-azino-bis (3-ethylbenzthiazoline-6-sulphonic acid) (ABTS) as substrate. Oxidation of ABTS was detected by following the absorbance at 420 nm (e420 nm =36000 M−1·cm−1) during 2 min with a spectrophotometer (Cary 60, Agilent Technologies, USA). The reaction mixture (1 mL) contained 10 µL of appropriately diluted enzyme sample, 890 µL of acetate buffer (100 mM pH 5.7) and 100 µL of 50mM ABTS in Milli Q® Water at 30°C. The ABTS was added to initiate the reaction. One unit (U) of laccase was defined as one micromole of substrate oxidized per minute in these described conditions.

* **Preparation**
An ABTS assay was conducted to measure the laccase activities. ABTS was dissolved in water according to the manufacturer’s recommendations. To find out if the laccases were active at all, the following filter paper test was performed. A colour change would indicate the laccase activity.

Filter paper test :

+ First, soak the filter paper in ABTS solution (2 mM ABTS, 100 mM phosphate-citrate buffer, pH 4)
+ Dry the paper at 60°C and add 10 µl of enzymes glycerol preps
+ Incubate at 30°C for 10 min

In addition, a 96-well plate assay was used to test the activity of the laccases. The experiments were done in two different pHs, pH 5 and pH 7, of phosphate-citrate buffer (100 mM). Absorbances were measured at A420 in one minute intervals with the Tecan M200 plate reader.

# Phage Display

* **Phage Helper preparation**

+ **Day 1**

- Thaw TG1 cells at 37°C : dilute cells in 100ml 2YT prewarmed at 37°C, shaking 240 rpm and are grow to OD600 0.5
- Add 8x1011 phages M13K07 helper phage in each → 30 min RT no shaking
- Centrifuge 10 min at 3500g
- Resuspend pellet in 300 ml 2YT + 50 µg/ml Kanamycin → 30°C ON shaking 250 rpm

+ **Day 2**

- Centrifuge culture 30 min at 12000g 15°C (sterilized falcon)
- Precipitate the phages in supernatant by adding 1/5 volume of (20% PEG8000 + 2.5M NaCl filtered) → 1h on ice- Centrifuge 15 min at 3000g 4°C
  - Resuspend phage pellets into 20 ml PBS sterile
  - Add 4 ml 20 % PEG8000, 2.5 M NaCl → 1h on ice
  - Centrifuge 15 min at 3000g 4°C
  - Resuspend pool phage pellet into 2 mL PBS (or more if 2 mL are not enough) (sterilized eppendorf)
  - Centrifuge 5 min at 15000g 4°C, put SN in fresh sterilized tube on ice
  - Centrifuge 5 min at 15000g 4°C, put SN in fresh tube on ice
  - Determine the phage titer : 1 OD260 = 3x1010 pfu/mL (lecture au Nanodrop, mode ADN)
  - Make several aliquots of 500 µL and stored them at -20°C

* **Phage’s bank**

+ **Day 1**

- 3x109 TG1/pHEN4 cells in 100 ml 2YT prewarmed at 37°C + 100 µg/mL Ampicilline + 2 % glucose → shaking 240 rpm and are grow to OD600 0.5
- Add 5x1011 phages M13K07 helper phage in each → 30 min RT no shaking
- Centrifuge 10 min at 3500g
- Resuspend pellet in 300 ml 2XTY + 50 µg/ml Kanamycin+ 100 µg/mL Ampicilline, 30°C ON shaking 250rpm

+ **Day 2**

- Centrifuge cultures 30 min at 12000g
- Precipitate the phages in supernatant by adding 1/5 volume of 20 % PEG8000 + 2.5 M NaCl → 1h on ice
- Centrifuge 15 min at 3000g 4°C
- Resuspend phage pellets into 20 ml PBS cold
- Add 4 ml 20 % PEG8000, 2.5 M NaCl → 30 min on ice
- Centrifuge 15 min at 3000g 4°C
- Resuspend pool phage pellet into 2 ml PBS cold (sterilized Eppendorf)
- Centrifuge 5 min at 15000g put in fresh sterilized tubes on ice
- Centrifuge 5 min at 15000g 4°C → put SN in fresh tube on ice
- Determine the phage titer : 1 OD260 = 3x1010 pfu/ml and put them at 4°C
- Coat 5 wells of a Nunc immunoplate MaxisorbTM surface with 100 µl coating buffer PBS and 5 wells with 100 µl of 100 µg/ml of antigen in coating buffer PBS at 4°C ON

* **Selection 1**

+ **Day 1**

Prepare 3 type of small Petri : ampi (16 for 1 antigen), kana (13 for 1 antigen), 1 without antibio- Place Polystyrene plastic and polyethylene plastic in a isopropanol solution (90 %) 30 min RT (don’t forget the negative controls without the plastic and with PBS solution instead)
- Culture of TG1 cells (8 μL) in 10 mL 2YT (in sterilized falcon) + 10 mL 2YT as a control
- Discard the isopropanol solution + rinse 4 times with 2mL PBS 0.05 % tween 20 and 4 times with 2 mL PBS
- Block with 1mL PBS 2 % BSA/well → 2h RT (1h can be enough)
- In the same time block 1mL phages in 1 mL PBS-BSA 2 % → 2h RT (1h can be enough)
- Discard blocking solution + rinse 4 times the plate with 2 mL PBS 0.05 % tween 20 and 4 times with 2 mL PBS
- Add 1 mL of blocked phages in each well
- Keep at room temperature 2h (after 2h put le plate at 4°C until the TG1 DO600 = 0,25)
- If TG1 DO600 > 0,25 dilute them to DO600 0,25
- Rinse 15 times the plate with 2 mL PBS 0.05 % tween 20 and 4 times with 2 mL PBS
- Elute the phages by adding 1 mL/well of 2 % BSA 0,2 M glycine HCl pH 2,5 → incubate for 10 min at RT
- Pool the 5 wells and neutralize with 1 mL 1 M Tris pH 7.2 (pipetting several times to pick up the phages)
**/!\From here, do the titration and the rest of the protocol at the same time**- Add 4 mL of TG1 cells at OD600 0.5 to the remaining 2 mL of phages (Ag well only) → 37°C 30 min no shaking (in the same time with the titration test)
- Add 18 mL 2YT + 100 μg/ml Amp + 2 % glucose → 37°C 15 min with shaking
- Add 1.6x10^10 M13K07 helper phage stock → RT 30min no shaking
- Centrifuge the cells at 4000 rpm for 15 min 4°C
- Resuspend the pellet in 300 mL 2YT + 100 μg/mL Ampicillin + 50 μL kanamycin → 30°C ON 250 rpm

- **••••• Titer of panning and control •••••**

* Prepare dilutions 10-2 to 10-7 for Ag and 10-2 to 10-4 for control (10 µL Ag + 90 µL 2YT for 10-2 tube and 10 µL Ag + 90 µL of 10x-1 dilution tube for 10x tube)
* Add 100µL of TG1 cells at OD600 0.5 to all the tubes → 37°C 30min no shaking
* Plate 50µL on the small Petri according the following table :

  | Antigen | | PBS (control) | |
  | --- | --- | --- | --- |
  | Amp | Kana | Amp | Kana |
  | 10-2 to 10-7 | 10-2 to 10-4 | 10-2 to 10-4 | 10-2 to 10-4 |

  * Plate 50µL of TG1 on an Petri + Amp, Petri+kana and Petri without antibio (TG1 has no antibio resistance so they technically can throw on no-antibio Petri) → Incubate 37°C ON

+ **Day 2**

All the Peg8000+NaCl and the PBS must be sterilized and stored at 4°C- If the TG1 control is ok, estimate the titer and the enrichment factor and keep the 6 Petri + amp Ag at 4°C
- Centrifuge cultures 30 min. at 11000g (distribute 50mL of culture per sterile falcon)
- Precipitate the phages in supernatant by adding 1/5 volume of 20 % PEG8000 + 2.5 M NaCl 1h on ice (40 mL of supernatant + 8 mL of PEG+NaCl per new sterile falcon) → 1h on ice
- Centrifuge 15 min 3000g 4°C
- Resuspend phage pellets into final 20 ml of cold PBS
- Add 4 ml 20 % PEG8000, 2.5 M NaCl → 30 min on ice
- Centrifuge 15 min 3000g 4°C
- Resuspend pool phage pellet into 2 ml PBS (in sterilized Eppendorf)
- Centrifuge 5 min at 15000g, put in fresh sterilized tubes on ice
- Centrifuge another time 5 min at 15000g, put in fresh sterilized tubes on ice and put SN in fresh sterilized tube on ice
- Determine the phage titer; 1 OD260 = 3.1010 pfu/mL
- Coat 5 wells of a Nunc immunoplate MaxisorbTM surface with 100 µl coating buffer PBS and 5 wells with 100 µl of 100 µg/ml of antigen in coating buffer PBS at 4°C ON

* **Selection 2**

+ Same protocol as Selection 1 but with the phages from selection 1
+ If the titration gives good results (more than 3log difference between Ag and PBS control) it’s useless to precipitate the phages. You can do the Screening by ELISA
+ If it’s not, do an 3rd round of selection

# ELISA screening

* **Day 1**

+ Pre-culture 96 clones in a 96-well DW 🡪 1 mL 2YT (or LB) + Amp (100 µg/mL) ON 37°C 200rpm
+ Do not forget to start the culture of the HHV(s) for the + and NR controls

* **Day 2**

+ **EXPRESSION**

- Start cultures of x clones (12, 24, 48 depending on the project) from the pre-culture in DW 24 well: 4 mL 2YT + Amp (100 µg/mL) + 0.1 % glucose + 60 µL of pre-culture 🡪 37°C 200 rpm
- Add 30 % glycerol to DW96, annotate (96 clones - antigen name - panning round number - date + DW map if required) and store at -80°C
- When 0.5 < OD600 < 0.8 (about 2h) induce cultures with 1 mM IPTG (prepare a stock solution 2YT + Amp (100 µg/mL) + 10 mM IPTG and add 400µL per well for induction) 🡪 ON 28°C 200 rpm

+ **ELISA**

- In a Nunc plate coater antigen (do not forget antigen for control(s)) and PBS (ctrl -): 100µL per well at 20µg/mL 🡪 ON 4°C

* **Day 3**

+ **TOTAL LYSIS**

- Centrifuge the DW 3000g 15 min 4°C
- Empty the supernatant and freeze the pellets at -20°C minimum 1h
- Thaw the pellets at RT for about 15 min ♦ Start washing the Nunc plate (ELISA) ♦
- Add 100 µL of 20 µg/mL PBS+DNase solution per 1mL of culture to each well (i.e. 400 µL of solution for 4 mL) 🡪 Shake 250 rpm RT minimum 30 min (more convenient to do it in the infors)
- Centrifuge 3000g 10 min 4°C
- Dispense supernatants into the rinsed and blocked Nunc plate, respecting the plate layout

+ **ELISA**

- Empty the plate and wash 3 times with 300 µL of PBS
- Block the plate with 300 µL of 2 % PBS-milk 🡪 2h 37°C
- Empty the plate and make 3 washes with 300 µL of PBS
- Dispense the supernatants into the plate
- Incubate ON at 4°C

* **Day 4**

+ Empty the plate and make 3 washes 300µL PBS+Tween 0.1 % then 3 x 300 µL PBS
+ Add 100 µL/well of Ac-mouse-anti-HA solution 1/500 in PBS-milk2% 🡪 1h at 37°C
+ Empty the plate and wash 3 times 300 µL PBS+Tween 0.1 % then 3 x 300 µL PBS
+ Add 100 µL/well of 1/5000 Ac-anti-mouse-HRP solution in PBS-milk 1 % 🡪 1h at 37°C
+ Empty plate and wash 3 times 300 µL PBS+Tween 0.1 % then 3 x 300 µL PBS
+ Develop with either : BM Blue POD Substrate (ROCHE) 100µL/well and block the reaction with 50 µL of 1 M H2SO4
+ Or make the reagent yourself: dissolve 1 tablet of TMB (Tetramethyl benzidine Sigma T5525-50TAB) in 100 µL DMSO, add 9.9 mL 100mM NaAc pH6.0 + 2 µL H2O2 then block the reaction with 50 µL 1M H2SO4 (/!\ calculate the volume of reaction solution needed before and adjust the volumes)
+ Read the OD450nm

# Killer digestion

When a vector is cloned with an insert some restriction site "disappears". Sometimes after the ligation reaction takes place, there may be contamination by empty plasmids (ones without insert). To get rid of this contamination, a killer digestion is performed, the aim of this digestion is to linearise the empty vectors by digesting the ligation mix by using one of the enzymes that recognize a restriction site that is still present on the multiple cloning site. In our case, if the vector was empty we could use Sac so it was the latter that was chosen to to linearise the vector (completely reducing the chances of transforming the cell since the vector is no longer circular). Following this digestion, we will find in the ligation medium the vector with its insert (in its circular form : absence of the Sac 1 site) and an empty linearised vector.

* **Digestion mixture**

+ 5 microLitre ligation volume (empty pet + PP pet + PE pet)
+ 1 microLitre of CutSmart Buffer
+ 0.5 microLitre of Bag 1
+ 3.5 microLitre H2O (QSP 10 microLitre)

Incubate at 65°C for 10 min or Incubate at room temperature (37°C) for 1 hour.

# Resin reuse

The HisLink™ resin and conditioned column can be used to purify proteins. More than once (usually two or three times), but it’s strongly recommended that the recycled resin is only used for the same protein.
To reuse a packed column, re-equilibrate the column with 20 volumes of binding buffer before reapplying the lysate.
Binding buffer: 100mM HEPES (pH 7.5) 10mM imidazole 500mM NaCl (for HQ-tagged proteins)

{% endblock %}

{% extends "layout.html" %}
{% block title %}Results{% endblock %}
{% block page\_content %}

# 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**).

![](https://static.igem.wiki/teams/4189/wiki/laccase/laccase1.jpg)

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

![](https://static.igem.wiki/teams/4189/wiki/laccase/laccase2.jpg)

**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 :

* 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.

![](https://static.igem.wiki/teams/4189/wiki/laccase/laccase3.jpg)

**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 :

* 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.

![](https://static.igem.wiki/teams/4189/wiki/laccase/laccase4.jpg)

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

![](https://static.igem.wiki/teams/4189/wiki/laccase/laccase5.jpg)

**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**).

![](https://static.igem.wiki/teams/4189/wiki/laccase/laccase6.jpg)

**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**).

![](https://static.igem.wiki/teams/4189/wiki/laccase/laccase7.jpg)

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

![](https://static.igem.wiki/teams/4189/wiki/laccase/laccase8.jpg)

**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.

![](https://static.igem.wiki/teams/4189/wiki/laccase/laccase9.jpg)

**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](https://2022.igem.wiki/aix-marseille/model).

# Peptides-GFP

To see our "Part collection", click [here](https://2022.igem.wiki/aix-marseille/part-collection).

|  | 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.

![](https://static.igem.wiki/teams/4189/wiki/phage-display/phage-display2.png)

**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.

![](https://static.igem.wiki/teams/4189/wiki/phage-display/phage-display3.png)

**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).

![](https://static.igem.wiki/teams/4189/wiki/phage-display/phage-display4.png)

**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.

![](https://static.igem.wiki/teams/4189/wiki/phage-display/phage-display5.jpg)

**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).

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