| 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. |
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.
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.
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 :
Waiting for our genblocks corresponding to the Fungal and bacterial laccase.
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.
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):
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.
Waiting for our genblocks corresponding to the Fungal and bacterial laccase.
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.
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.
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.
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.
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.
Pause/interruption of the design of a potential upgraded laccase to help in other experiments.
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.
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.
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.
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.
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.
No progress cause of vacations and public holidays
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.
Pause/interruption of the design of a potential upgraded laccase to help in other experiments.
We did a basic 6his Purification with pET expression of PE1C6.
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.
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.
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 :
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.
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.
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.
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 :
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.
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.
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.
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.
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.
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.
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.
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).
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.
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.
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)