Official first day. However, not everyone is in London. We are still sorting out funding.

Edward and Alberto arrived in London. Miro sent out sponsorship emails. Georg and Alberto chased Baldwin for CDT studentships.

Alisdair, Sonia, Edward, Alberto, Kevin and Georg meet at 10 am on campus. In the morning we worked on sending out sponsorship emails. We also sent out e-mails to the Head of Department (and president of the Biochemical Society) Anne Dell for travel grants. Kevin is trying to contact the BBSRC (since it sponsored the Oxford team in 2017). Ismael arrives to say hi at 12. Lunch in JCR. In the afternoon we set out more sponsorship requests. We also went to the enterprise lab to ask for more funding. Our design student Edward has started working on the logo.

Slightly positive news from the Enterprise Lab for funding. We discussed what to discuss for tomorrow’s meeting with Oscar Ces. We planned steps in wet lab and dry lab. First name idea: Nano DIPs (DNA Imperial/Intelligent Pores). The final year biochemists got their transcripts today. Alisdair downloaded Cadnano. Eddie is working on the logo. The raspberry pie arrives.

Meeting with Oscar, 9am (white city). Apologies from Miro: I would like to Skype in. Present: Sonia, Ismael, Alisdair, Georg, Alberto, Edward, Kevin, Oscar Ces, David Miller We discussed logistics in the labs and feasibility of experiments. He suggested us to use Kanban boards to be efficient. It is essentially a highly organised japanese post-it system. We outlined some funding strategies (e.g. more targeted emails to just 10-15 companies and copying him in). At 10 we met with David. He suggested to make a list of things we need to start running experiments. The wet lab is going to replicate the Burns, Reifer and Howorka (2016) paper as a first sanity check. In the meantime, the dry lab (Alisdair) familiarises with Cadnano. Lunch in a street food market in White City. With IDT you can order DNA sequences with cholesterol at the 3’-ends, so they can attach to membranes. At 5 everyone goes home.

Sonia, Eduard, Kevin and Alberto meet at the hackspace in the morning. Georg, Alisdair and Miro are working remotely. No great news regarding sponsorhips. We tried to chase Baldwin but he was elusive. Sonia and Alberto made a protocol for making liposomes filled with fluorophores. We need an extruder to make multilamellar liposomes into unilamellar (just one bilayer). Our first logo is now online (https://nanodips.github.io/home/).

There is the chemical biology symposium in White City today. Apperently Stefan Howorka is talking there about DNA nanopores. We managed to sneak in at the conference asd listen to the lecture. Some thoughts about DNA Nanopores: "What is the benefit of transporting single DNA molecules through tiny holes ? DNA sequencing. Biosensing.Detecting analytes is possible using ON/OFF switch approach (not reversible). This can be adapted. to create a protein sensitive pore. The presence of a protein lead to specific signal. These DNA pores are completely new. What are the properties ? Not as stable as the protein pores… more wavy and flexible. This is something that needs to be addressed" (Howorka, 2019). Meeting with David at ICAH at 5

Meeting in White city in the morning. Present: Albi, Georg, Sonia, Kevin, Edward. Miro is working from Home. Positive news from the Bioeng department.Massive brainstorming day. We came up with the key-Tree idea. DNA keys are displayed to the surface of the liposome via a peptide linker. The peptide contains a recognition sequence for the MMP7 protease, which is secreted by metastasising cancer cells. Thus metastatic cells can trigger the opening of the pore and the release of a chemotherapy drug. We talked with Ryan about the issue with access to hackspace. The website is under construction. The protocols have been reviewed and approved.

Good news from the sponsorship front. The CDT in BioDesign Engineering announced that it will fund up to 8 6-week summer bursaries for undergraduate students (also final year students). Now we need to generate momentum and follow up with the Head of Department of Bioengineering. Also the Enterprise Lab is giving us 2ks. We can start buying flights already perhaps. In the afternoon, meeting at the Hackspace for the daily meeting with David. Prepare for meeting with Oscar tomorrow.

At 9, meeting with Oscar and David at the Hackspace. Things discussed: Novel idea: DNA key-tree. Cancer-specific proteases release keys. Maybe use trypsin instead of the hard-to get MMP7. Update about BioDesign CDT bursaries. He suggested to design every experiment as a controllable separate step. We need a lot of DNA (IDT !!!). Probably we can get more funding from the Hackspace. We need to be careful when using the extruder to wach out fluorescent stuff. Introduced Kevin to the world of Github Desktop. Eddie spilled water on himself and he is working on the vortex animation. Alisdair is working on cadnano. The IDT quote has been sent to David, hopefully DNA arrives soon.

Sonia, Kevin, Albi, Georg and Alisdair met at the Hackspace in the afternoon. Miro is working from Slovakia. Eddi is coming back to London from Austria on wednesday. Wrote other follow up e-mails. Update with key trees. Daily meeting with David at 5.

Everyone worked remotely. We sent the IDT quote to David. Laid down the basic architecture for the website. We are constructing it from scratch (no template, no nothing) in Atom now. We are still waiting for the extruder to arrive. As a result, the wet lab is inactive at the moment. Hopefully next week we can start making liposomes.

We ordered flights from London Heathrow to San Francisco. We are leaving on wednesday 23rd of October. Thank you Enterprise Lab ! The Jamboree is on the weekend. Then we will fly back on Tuesday. Apart from Georg and Eddi, who are going to stay in the USA until November. The IDT order has been received, so hopefully in a week we can start assembling some DNA pores. Eddi lost his flight.

Meeting in the Hackspace. We ate falafel wraps for lunch. Everyone is working on the website today. Eddi arrives in White City after lunch. Everyone is working on the website. Everyone is learning HTML and CSS. 49 years ago the Queen's had their first gig ever (at Imperial).

Meeting with Oscar and David at 9 am in the challenge room. The life scientists received the CDT bursaries forms. UROP forms have been signed. Oscar suggests to go to Anne Dell to extend our UROP. Otherwise we can only work 6 weeks in the lab. We get paid per hours, so we need to log the hours we work. We can only work 35 hours per week apparently. Oscar says this is a good thing, at least for our mental health. IDT order has been confirmed. Ismael suggests to chase IDT, as they might be very busy with the free IDT order. We could also ask the help from the Foundry. Let's write an e-mail and cc Oscar to the Foundry. It would be good to contact Stefan Howorka. However, this would count as discolusure to someone outside of the college. Maybe we should go to the Enterprise Lab to discuss pateting things. But should we think about patenting before the jamboree or just enjoy the moment [cit. O.C.]?

Meeting at the Hackspace in the morning. Present: Albi, Georg, Sonia, Edward and Kevin. Today is the first day in the wet lab, finally. It took some time to understant where things are in the hackspace. It is not as equipped as the labs in South Kensington. But it is very cool as it has that DIY feel. We dissolved phospatidylcholine in chloroform. The website front is progressing quickly.

Meeting in the morning at the hackspace. The website front proceeds, with Edward working on the design and Georg who is in his HTML loop. In the wet lab, Sonia and Alberto dissolved DOPE and DOPC in chloroforom as decribed by Howorka and let the chloroform evaporate overnight in the fumehood at room temperature. Hansa is here to help with the liposome preparation. We had lunch in Westfield. Since tomorrow is the last day when we can claim back some money from the Hackspace, we paid the BIOMOD registration fees and booked the hotel in San Francisco (the Marriot). Call with Miro for updates.

Meeting at the Hackspace at 11. It is an insanely hot day. We checked the lipid cake at 12 am. It was not a dry cake yet, but still a liquid solution. We asked David for help. He suggested to rotate the flask or pump some nitrogen inside. We tried both. Using a stream of nitrogen we managed to evaporate all the chloroform. Lunch at Westfield (but some people still had falafel wraps). Alisdair and Alberto met Tom Ouldridge, who gave really helpful suggestions regarding the key tree. Maybe now we could do a key kyte. Kevin sent all the invoices to the Hackspace.

Meeting with Oscar at 9, as tomorrow he is going on a holiday. The fridge broke during the night. The DNA from IDT arrives. In theory tomorrow we could already replicate the Howorka paper.

Very hot day. Tha AC broke. It's 32 C in the lab. While the sephadex gel is in the oven, we go for some drinks at 5 pm (it is friday at the end). Found a new bar that gives 25% discount to Imperial students. Georg wants to replicate a Nature paper at 5 pm on a friday. Everyone else disagrees. We tried to hack gel filtration without proper sephadex columns (we used syringes as columns and filter supports from the extruder as membranes).It was a disaster. Everything was dripping. We need proper filters. Sephadex 1 - Nanodips 0. In the dry lab, Alisdair identified the three toehold regions in the lock. That means we have to hide all three with 3-antikeys. That makes the design more complex than expected. In the meantime, Eddi is making progress with mMAYA. He modelled a DNA barrel with 6 cylinders embedded on a curved lipidic bilayer.

Meeting at 10:30 at the Hackspace. Present: Sonia, Hansa, Georg, Alberto, Eddi, Alisdair. Succesful day in the wet lab. What is great about using liposomes and DNA is the efficiency of the experiments. In less than 6 hours today we made fresh liposomes and incapsulated calcein inside. If we had to use cells we would still be waiting for them to grow. When a strong detergent (Triton-X100) is added in the solution of liposome, a twofold increase in fluorescence is observed. Without Triton-X the fluorescence of the liposome solution is similar to the blank (sucrose buffer). This means that calcein is encapsulated in the liposome at a self-quenching concentration of >60 mM. We left the solution of liposome in the plate reader overnight to investigate the leakage of fluorophore from the LUVs.

In the morning, we went to South Kensington to find a DLS machine and assess the quality of the liposomes from yesterday. We found a helpful postdoc in Freemont's lab who let us use the DLS machine. Great news: we have a very uniform distribution of vesicles, with median size of 67 nm. This is good as it means that we can make good LUVs in a day at the Biohackspace. In the dry lab, the key yte is being designed. There is now a pen and paper design. We need to add an extra layer of DNA to block all the three toeholds. At least the number of pepetide-DNA conkugates now is always one. We need to understant the chemistry to make it possible now. Today the aim is to replicate the Howorka paper. Bad News: the thermocycler is broken. That makes it hard to anneal the oligonucleotides for 2 min at 95 C to assemble the DNA nanopores. We could use a water bath, but since annealing is sensitive to changes in temperatures we decide not to attempt the experiment. Tomorrow we will go again to South Kensington to use a thermocycler. We need to make a video as on friday we will take over Imperial College's instagram account.

Today we finally replicate the Howorka paper. Beer at the Blue Moon Pub (the union of white city) at 5 pm, while waiting for the gel. We checked the plate reader results after 30 min. No difference in the wells with just liposomes, liposomes + pores + key.

Time to replicate Howorka again. Perhaps the negative charges (there are 5 carboxylic groups) in calcein do not allow it to pass through the DNA pore (which is negative as well due to the phosphate groups.) Let's try with Rhodamine (less negative). We start to wonder why in the original paper they specifically used sulforhodamine. Is it because it was the only thing they had in the lab or is it because it is the only molecule that trepasses the pores ?

Today we take over Imperial College's instagrams account (with 55k followers). We also have to replicate Howorka again. Sonia made nice sketches of the team members. In the supplementary information of Burns et al 2017, they show that the liposomes are larger than 100 nm. Indeed, with the pore size of the extruder we were actually making SUVs instead of LUVs.

We extruded with filters of pore size = 200 nm. Plate reader results look promising. Miro leaves the core team. Welcome Shabz, who studies Physics. Sonia, Georg and Hansa went to the cinema.

Meet at the Hackspace at 12 am. Present: Sonia, Albi, Hansa, Alisdair. Kevin and Shabz are working from home. Georg and Eddi are in Austria for holidays. We checked the plate reader results from friday morning. In the wet lab: agarose gel for cofirming pore assembly. Ordered more sephadex and sulforhodamine. The gel confirms the we assembled the nanopores from the oligos. Now let's prove that we can incorporate them in lipid bilayers and tranport molecular coargoes across them. Alisdair is proceeding at speed in the dry lab. The sequences for the key kyte are ready. We asked biomer if they can synthetise chemically a peptide-DNA conjugate. We received the claims back from the Hackspace.

Today was an office day. In the wet lab: waiting for sulforhodamine to arrive. Planned tomorrow's plate reader experiment. To test: effect of assembling LUVs with different concentration of DNA nanopores. Drafted e-mails to deans.

Plate reader experiment with sulforhodamine. Added nanopores after extrusion. Bad news from biomers regarding the synthesis of the key kyte: very difficult (and expensive) to synthesise it. Sent e-mails to deans.

The plate reader does not show SRB leakage. Are we sure that the nanopores are embedding in the membranes? Today we tried to incorporate the DNA nanopores not after extrusion but in the initial step. Perhaps the cholesters in the nanopores clump together due to hydrophobic effects (to make the famous clusterfuck). Mixing them with methanol and chloroform from the beginning might result in their embedding in the membrane.

Not a very productive friday. Georg and Eddi are still in Austria. Sonia and Albi are not in today. Kevin and Hansa made a lot of buffers. Alisdair designed toehold hairpins for restriciton enzymes recognition sites.

Meeting at the Hackspace at 11. Present: Sonia, Hansa, Albi, Alisdair, Kevin. It is a very rainy and cold day. However it is bright in the wet lab. We repeated the SRB leakage experiment with nanopores closed + key. It looks like it's working. There is leakage of SRB only in LUVs + NP + key. Nothing in mismatch. However, not everything is going out from the liposomes. We need to calculate the ration nanopore to liposome ration to investigate why. Good news also in the sponsorship form: the Head of Departmend of Bieongineering is giving us 2000£. Alisdair is finalising the sequences for the key kyte. We drafted questions for tomorrows's meeting with Burns (the first author of the DNA nanopore paper ). Kevin sent the application to Vertex. Eid Mubarak to everyone (especially to Shabz).

Promising results in the wet lab. We can confidently say that we succesfully replicated the Howorka paper. Also, interestingly the pore + mismatch key is leass leaky than without anything. Maybe we can use the mismatch key as a sort of pore plug. To celebrate we had lunch at the Athenian. At 2 pm, we skyped with Jonathan Burn, the first autho of the paper. He was happy that we are working on his research and that we were abe to replicate the paper. He suggested to perfrom a gel shift assay. If we want some cool pictures and date we should use Giant Unilamellar Vesicles (GUVs). We are officially out of DNA. Ordered more DNA. Now we can read papers and plan next experiments until monday.

Early meeting in the Hackspace. The wet lab team meets in the challenge room for the Journal Club. Each one presents a paper that Burns suggested and summarises the main findings to the others. Alisdair is trying to remove toehold regions to further simplify the DNA sequences.

Day off. Nothing to declare.

Meeting with Oscar at 9 am. We showed him the results from the wet lab. Results are tantalising but we should replicate the experiment again (with 3 replicates per sample). It is important to compare the results the vesicles with nanopores with just vesicles without nanopores and assess leakiness. If we want to make good pictures we should make GUVs. However we cannot make GUVs by extrusion. We can make them with phase-transfer or electroporation. These are definetely easier than making a microfluidic chip. We could also order fluorecent lipids for graphical purposes. In the dry lab: we ordered the half kyte with fluorophores for testing and brainstormed alternative design ideas for streamlining the key kyte. Today it was also Pizza Day. Alisdair, Shabz and Hansa braved the rain, credit card malfunctions, the frustration over the hackspace not being in the catchment area of ANY dominos outlet.... and argued over the fallacies of vegetable toppies vs cheese only.

Present: Sonia, Hansa, Georg, Alisdair, Eddy. In the wet lab: we are still waiting for DNA to arrive. Hansa and Sonia prepared a lot of megamixes. Mr Entropy (a soft and furry purple ball that is the mascotte of the plate reader is lost. Fortunately we replaced him with another Mr Entropy. New micropipettes were delivered. Sonia did not lose the chance to make a new unboxing video. The Kanban is back.

In the wet lab, Sonia and Hansa were very proud for figuring out the maths of the lipids. We realised that we only had 2 vials of the pre dried lipids left. So we tried to add everything inside in one go and evaporate it. However we noticed that the vials of lipids were frozen (or dried). Perhaps all the chloroform evaporated. So we opened new vials. We redid calculation for stock and working lipid concentration. Apparently we were 3 orders of magnitues off (milli instead of micro). Apparently we must have had really fat vesicles. We therefore updated the protocols on Benchling with new concentrations. Now the lipids rations are very elegnt (3:7). We did serial dilutions of open nanopores. We set up a new experiment assessing different concentrations of open nanopores. We made record vesicles: 6 fold increase in fluorescence upon addition of triton. There must be a lot of sulforhodamine inside. Set up a plate reader experiment overnight. Georg and Shabz made a machine that maked Giant Unilamellar Veisvles (GUVs). It is essentially a square-wave voltammetery "thingy pingy" [G.W.]. Alisdair finalised sequences ready for a new order and to test peptide linker. Eddy worked on mMAYA (nothing new under the sun) and showed a cool video of the inside of a vein. You can see red and white blood cells flowing into a channel surrounded by cells.

As a first thing in the morning we checked the overnight results in the plate reader. Most of sulforhodamine leaked out before the plate reader could detect it. However a sample (the one with picomolar concentrations of DNA) has good curves of SRB release. Apperently less is better. There was a delegation of people in the Hackspace. They visited our Lab. Hansa explained them our project with an analogy with shoes (?!). The whole team assembles in the challenge room to decide on the storyboard for the video. We will start by highlighting the big problem (the need for more targeted drug delivery) and then narrow it down specifically to metastatic cancer and then present our solution. The style of the video shall be clean, simple, 3D and professional. Ismael joins us at the hackspace. He sneezed 4 times in a row (instead of 3). We went for lunch at "Herman Ze German". Sonia had a vegan sausage. The italian prime minister resigned. After lunch: ordered chemicals for linking DNA with peptides. Sigma is like Amazon for chemicals. We ordered DNA with extra amine residues at the 3'end. They will attach to a heterobifunctional linker (SMCC), which can react with the cysteines molecules at the edges of peptides. At 5 Albi, Sonia, Georg, Eddy and Shabz call it at day. Hansa and Alisdair keep on hacking at the Hackspace.

We are expecting DNA this morning at 10:30. If DNA arrives the 9-well plate will be an army of liposomes and nanopores. Turns out that the IDT parcel was in the Hackspace since yeasterday. Sonia brought bitter matcha cookies. She offered them to th security officer (Aifon, or as some people call him "the spec controller"). He refused them. Sonia made a really funny unboxing video. When looking at the tubes we realised that they only sent us 5 tubes. However to make a nanopore we need at least 6 oligos. Apparently they only sent us oligos without modifications. Cholesterols and fluorophores might take longer to synthesise. Thus the wet lab is left without any experiment. Sadly, we retunr to the office and work the hole day on the website. We had lunch under a tree. Somehow Eddy found the lonely tree. The wet lab team made a first draft for the protocols. Eddy is working on the video and the design for the build page. Hansa and Albi analysed data for the vesicles without any nanopore. Sonia wrote and proof-read the build and intro page. Georg is coding the backbone for the build page. Alisdair is working on the sequences and the upstream circuit.

Meeting with Oscar at 9 a.m. We showed him the data of vesicles without nanopores. Judging by the slope of the curve, he agrees that they are not leaky. Oscar suggests that the mismatch key acts as a molecular poreplug (or buttplug). Pehaps this is why it is less leaky. For the triton data he suggests to add a break out point in the graph (with data without triton on the left side.) We had croissants and coffee at the cafè. Talked about the lord of the ring (nothing new under the sun). We have fans on Instagrams for Sonia's unboxing videos. Domino's Pizza for lunch. Sonia, Albi and Hansa weep on writing protocols for the build page. Georg is making a lot of accordions. Alisdair is reading papers and thinking about the circuit and sequences. He also applied to a grant for Bioengineering. Oscar needs to write a reference for that. Sonia is making more sketches. Kevins went to the education office to have updates regrding the salaries. Apparently we need to wit until the 24th of September before receiving the money.

Today is Bank Holiday. Day off for everyone.

Everyone working on the website. We drafted a script for the video.

Everyone working on the website.

We came early in the morning to collect the DNA parcel. Hansa, Shabz and Alisdair had another philosophical conversation over Margherita Pizza and deliveroo scams. Hansa, Albi and Kevin brainstormed about the introduction and build page.

In the wet lab, Hansa, Sonia and Albi the classic liposome experiment. Alisdair, Georg and Shabz met with Tom Ouldridge to discuss about the modelling. He said that the model that we devised is reasonable. He criticised one assumption that we made. Ismael brought mead after the meeding. It was stupendous. At 5 we went to the Blue Moon. We had Espresso Martinis while waiting for the gel to settle.

Meeting at the Hackspace in the morning. Present: Sonia (she is back, yay!), Hansa, Gerog, Albi, Kevin, Eddy. It was an extremely unsuccesfull day in the lab. The gel did not work. The plate reader for the key kyte looks promising. The sephadex columns did not even settle. We could not even incorporate SRB inside the liposomes. Perhaps the SRB solution is "expired". We did it more than 3 weeks ago. We ran out of lipids. We had lunch at the Athenian. The security officer said that we are legends. We met with David and ordered more lipids and more reagens for the nanopocs. At 6pm we went to Kentish town to get the DNA from the UPS collection point.

In the wet lab Sonia, Albi and Kevin worked on the key kyte. We incubated it at 37°C in the plate reader. Sonia made a new unboxing video (new lipids arrived). When the two strands disanneal the Black Hole Quencher should leave and the signal for fluorescence for fluorescein should increase. We observed a significant increase in fluorescence after 40 min incubation at the lowest concentration of DNA. We had lunch at Herman ze German and talked about opium, microdosing and Kidzania. In the afternoon, the wet lab team tries to replicate the good key kyte results (while watching friends in the background). Georg finished the Build page. Eddy is working on the video and changed the introduction. At 4 everyone discusses the video with Eddy. What is the name of the man with cancer ? Georg proposes Biotod. We ran out of Smaug (the restriction enzyme SmaI).

In the wet lab, we replicated the key kyte plate reader experiment with decreasing concentrations of DNA. We receiveid more Smaug so we can replicate again the key kyte experiment. We also watched How I met Your Mother while filling the many wells of the microplate. We had falafel (with hummus on top as a gelafel) for lunch. Shabz got curry goat instead. Hansa had "The Greedy" from the Athenian. During lunch, we talked about squatters and their rights. We checked the results from the key kyte. No increase in fluorescence. So we are not sure if the experiment from yesterday was just a fluck. We will try again tomorrow with new concentrations. We are shooting in the dark. After lunch, we hydrated new liposomes and performed the classic Howorka experiment with nanopores open, nanopores closed and empty liposomes. We also prepared a very thick agarose gel (4%). This would hopefully trap all the short oligos from running out of the gel. Shabz solved PDE equations. The first one succesfully. The second one not succesfully. He is now working on calculating the binding energy of the toehold with the neares neighbor model. Alisdair is still sorting out his car situation. Albi made 10x TAE buffer and used is as if it was 1x. Perhps that's why we didn't see anything in the gel. In the website front, Georg made the navbar responsive. Georg is also sad because of the results from the key kyte. Eddy worked on Maya and smoked more cigarettes. He then had to run away as he had an hairdresser appointment. Sonia will go home by foot as she suspended her own card for no reason.

Performed the key kyte digestion experiment again. We incubated 1 microgram key kyte at 25°C in the plate reader. However, there were no differences between test and control. We checked the results from yeasterday liposome experiment. LUVs, LUVs + nanopore closed and open both show a decrease in fluorescence overtime. We discussed possible hypothesis for this. Perhaps we did not heat them up and chloroform not evaporated interfered with the elution. We watched "lord of the ring: the return of the king" while pipetting. Had lunch at Waka (with Emma as well). Eddy is very angry with Maya, which keeps on quitting "unexpectedly". In the afternoon, we casted another 4% agarose gel ti visualise the digested bands of the key kyte. Finally we can see the bands of the key kyte. However there does not seem to be much difference between the digested and not digested. Probably then the probelm is the sequence itself. We ordered another key kyte design and quantified the concentration of ssDNA and dsDNA with the nanodrop.

In the morning, the wet lab people started the Howorka experiment (again). We checked the gel from yesterday. The oligos were more separated. However the bands are too faint to confidently observe anydifference between test and control. We were very efficient and managed to extrude even before lunch. We ordered pizza from Mariano for lunch. We had lunch while watching How I met Your Mother. After lunch we got back to the lab, eluted and sent the dope army in a mission to the plate reader overnight. In the meantime, we finished the Return of the King. Eddy and Georg are viewing an apartment. Albi lost his phone.

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Final official day of BIOMOD.