Monday

First day of iGEM 2018. Everyone meets. We took the first picture in queen's tower .

Tuesday

Brainstorming day. We met our PhD supervisors: Ismael and Amrit. We also met Rodrigo, one of our two PI supervisors.

Wednesday

Lots of brainstorming again. We met Will Wright, the European iGEM ambassador. We attended an Outreach event organised by Nico McCarty. We presented to some high school kids what iGEM and synbio is. We also helped the students with some bacterial transformation tutorial (for Biopanting with GFP).

Thursday

Brainstorming is intense. So far three candidate ideas are held by the general team consensus: electronic control of patterning, biofilm lithography, control of heterocyst-cell differentiation in E. coli, Catalytic Biofilms (?!), production of quantum dots, game theory simulation, et cetera... First team trip at the union.

Friday

Breakfast reception with the High School students from wednesday in the microbiology building . Brainstorming again.

Monday

We visited for the first time the lab we are going to work in during the summer. It's the Phillip Lab in RCS floor 1. Thank you Eppendorf for the PCR thermocycler, centrifuge and incubator. Team PicNic in Shepherd's Bush with Rodrigo and then visit to OpenCell, where there are supercool labs inside shipping tanks.

Tuesday

Presentation of the main ideas to the Centre of Synthetic Biology. Positive feedback for the electronic control of patterning idea. The game theory simulation idea has been killed by the feedback from the PhD students (sorry Shiv...).

Wednesday

We met Alice, an artist from RCA, who crystallises sweat from people and makes fashionable pieces of clothing with it (?!). She helped us brainstorming by promoting a discussion outside of our biochemical comfort zone.

Thursday

Shiv, Alberto, Josh and Thom are in Oxford for the iGEM Meetup. The rest of the team remained in London to work on ideas and to wait for some artists from the RCA. Unfortunately, the artists never turned up. Are we really that intimidating ?!

Friday

As Thursday. We also met the iGEM president: Randy Rettberg.

Monday

Week three has just started. Shiv, Alberto, Josh and Thom are back from the Oxford meetup. We met Tom Ouldridge in the morning, who returned from holiday. First group problem: Lidia decided to leave the group. Everyone hopes she is coming back. Let's remember that amazing team dynamics are reached with "Radical Truth and Radical Transparency". A wild Prof. Kitney appears around lunchtime: it is the first time we see him officially. We talked about the idea of patterning. He was very chill and supportive. He also proposed to have two separate wikis: a public one with limited info (so that other teams cannot copy us) and a private full one, to be published right before the freeze. He also told us when he went to holiday with his wife in China (?!). Quote of the day: "you may have heard of me." Rodrigo and Tom Ouldridge came into our brainstorming room after lunch and we presented the main idea: "PixCell: electronic control of biopatterning". It was a constructive discussion. It's time to think about simple backup plans. What if the agar is not conductive enough and nothing works ? Rodrigo proposed that we could solve this by placing electrodes in solution and control the temporality of gene expression (perhaps of co-cultures in bioreactores to split biosynthetic pathways between different communities and prevent burden)

Tuesday

Presentation of PixCell to the centre. Kitney liked it. He suggested to think about application... think about the "so what..". Positive feedbacks from Tom Ellis, start thinking about the big picture: after all we want to control bacteria electronically: cybergenetics? PixCell has officially started. The game is on. Tom Ouldridge held a brainstorming session and we assigned roles and things to do. Lidia is back, stronger than ever. She is going to be our amazing group leader, the glue... Agenda for tomorrow: Luis: meet with Pascal, redox modelling Will: meet with Pascal, redox modelling, electric circuits Yutong: meet the graphic designer, design circuits and identify what we need to order Josh: design circuits Diellza: design circuits Thomas: human practice strategy in the afternoon and wet lab strategy Alberto: human practice strategy and join wet lab strategy in the morning and catalogue parts in silico Siwat: human practice strategy in the afternoon and wet lab stategy Lidia: design circuits and model transcription Shiv: unassigned yet Trip at the Union to celebrate the start of PixcCell.

Wednesday

In the morning the wet lab team is bulding an inventory of parts and consumables we need. We are probably get SoxR and pSoxS with a genomic expansion (as these are endogenous in E. coli). We are also making a list of PIs and labs here at Imperial who could give us some useful reagents (such as Assembly kits). Dry lab team are full on modelling. Apparently constructing the electrode is not as complex as we expected. The graphic designer friend of yutong from CSM (virginia) had lunch with us in JCR and we discussed about our project. She is going to make our logo and help us with the aesthetics of everything. In the afternooon we constructed the inventory of materials, consumable etc... Hopefully we have the primers for the genomic expansion of SoxR by monday and we can start to pipette something ! Siwat danced Pen Pineapple Apple Pen. At 5 we had the meeting of the day with PhDs and PIs: everyone summarises the findings and achievement of the day and plan the agenda for tomorrow. Rodrigo and Tom are positive with the day. What about a team dynamics interactome ? Agenda for tomorrow: Luis: autoclave training, e-mail for ComSol, Redox modelling Will: autoclave training, redox modelling Yutong: autoclave training, contact merchandinsing, parameters for modelling Josh: autoclave training, ask Rodrigo for Cell Free, modelling Diellza: autoclave training, authorization request for ordering, DNA design Thomas: autoclave training, DNA parts design Alberto: autoclave training, modelling parameters Siwat: autoclave training, modelling parameters Lidia: autoclave training, Kirsten, modelling transcription Shiv: autoclave training, authorization request for ordering Amrit: risk assessment to order FeCN and PyO.

Thursday

In the morning a representative from BMG Labtech came in the lab. We got a microplate reader from them. We did a tutorial to learn how to use it. Lunch break in queen's lawn Luis, Will, Aberto and Siwat in the dry lab, attempting to model and find parameters (not very succesful) The wet lab team remains in RSM 1.47 designing primers and parts. Full inventory is done. Tutorial for microplate reader is on the google drive. Rodrigo: why don't you use chromoproteins istead of GFP ? At 17: lab meeting. The wet lab team completed the invetory of lab material to order. DNA sequences also have been designed. In the dry lab, Will and Luis continued working with COMSOL for the modelling of the potential at the electrode surface. Alberto and Siwat looked for parameters required for redox modelling of Fe(CN)6 and Pyocyanyn. Agenda for tomorrow: everyone risk assessement turotorial in the mornng (Phillips lab) Luis: model, plot concentration vs volum Will: model, PCB design Yutong: finish primer design, IDT order, order DJ901, talk to BMG representative for toxins on plate reader Josh: finish primer design, IDT order, order DJ901 Diellza: finish primer design, IDT order, order DJ901, Eppendorf Thomas: go to hackspace, look for collaborations and feedbacks. Alberto: list of chemists @ imperial + visit electrochemistry labs to find about redox reaction entropy (ferrocyanide) + look for parameters Siwat: list of computing/physiscs @imperial that use COMSOL, look for parameter Lidia: primer design Shiv: transcription/translation modelling, contact artists Ismael: risk assesment, iGEM kit Amrit: holidays

Friday

In the morning everybody had the safety induction in the Phillips lab with the lab manager. No students are allowed alone in the lab after 5 pm. Wet lab: filled safety forms. Designed primers. Protocols have been added here on benchling. Rodrigo apparently has the GFP in the plasmid we wanted. Yutong talked to the plate reader guy about the use of pyocyanin on their plate reader. We filled the biosafety BIO1 safety form (for the overall project). Then we need a COSHH for pyocyanyn. SOP form was not completed. We need to fill it in with our protocols. Dry lab: exercised on COMSOL again. Alberto spoke with Dr. Andrea Fantuzzi (electrochemist in 7th floor in SECB), he liked the setup but he's worried about the material of the electrode (silver can react with Cl to form AgCL which causes cytotoxicity). However, overall he seemed excited. Free cell free stuff from TXTL cell free.

Monday

In the morning, we completed the BIO1 form and all the relevant SOP forms for biosafety. The dry lab team made progresses in the modelling. Basic deterministic TXTL is running, reasonably simplified. Start working on a stochastic model as well. We are still waiting for the electrochemistry package in COMSOL. Wet lab managed to get hold to a new vector that converts Golden gate into BioBricks (developed by Marko, a researcher in Geoff Baldwin's Lab). We have ordered Pyocyanin, petri dishes, glycerol, ethanol, and most importantly primers. Alberto and Thomas spoke with Charlie Keyzor (president of SynBIC) and we are going to have part of the SynBIC stand on Fresher's Fair to showcase our iGEM project. Yutong's friend is deisgning a pixcell mascot. At 5 meeting as usual. Apparently we did not fill all the forms to receive all the payments. Tom updates us on the plan for the Jamboree. Agenda for tomorrow: everybody at 10 a.m. in Phillips lab for nanodrop tutorial Luis: contact comsol for sponsor Will: arduino Yutong: not in the morning (bank), go through all the wikis and see if there's a team workig on similar project (for collab) Josh: collect expression vectors, miniprep/glycerol stock Diellza: dry lab autoclave, anitibotic solution Thomas: meet SciCom guy Alberto: meet SciCom guy, e-mail bioethicisic fro European commission Siwat: meet SciCom guy Lidia: e-mail Sara Biol, Molly Stevens and Collab presentation Shiv: find hardware teams

Tuesday

Today was the first day in the wet lab. In the mornig we had the NanoDrop turorial. Alberto, Thomas and Siwat had the meeting with Dr. Sternberg (senior lectures in science communication) who gave helpful advices on how to comunicate complex foundational technology. Wet lab team and dry lab team remained in the Phillips lab. We then headed off to farmer's market for lunch. In the afternoon we prepared the antibiotics aliquot, the LB agar and the solvent solutions. We also picked up the MG1655 strain lines and Turbocompetent cells. from Tom Ellis -80 freezer. At 5 pm meeting in the lab with Amrit and Tom.

Wednesday

In the morning we received the parcel with pyocyanin and also the NEB free stuff. We did everything in the checklist. We make Agar-LB plates. We also resurrected the old plasmid (pBR322) and spreaded the SoxR-containing E. coli strain (MG1655) into plates. Primers have been shipped. Dry lab: RBS and promoter strength does not seem to affect the system. We thought that the general theme of our human practice is communication (exchange of information): Communication between scientists and general public. Communication between individual cells to form complex patterns Communication between individuals within a team Communication between machines and organisms (computers connected to bacteria) After lunch, Alberto and Shiv met two artsts from UAL and presented PixCell and proposed the Art exhibition. We received the confimation e-mail for the jamboree. At 5pm usual meeting with T.O. and Amrit.

Thursday

One of the hottest day so far (around 35 °C). First failures in the lab: our GFP overnight cultures did not grow. Primers haven't arrived yet and so we could not do much more. Also we found out that the sterile loops are not autoclavable... we found them melted in the morning. The modelers are trying to see if they can code the electrochemical model with MATLAB, as COMSOL people did not reply yet.

Friday

We finally received the primers and performed colony PCR to extract SoxR from an old strain, a sample of which was in some freezers in Tom Ellis lab. Shiv, Lidia and Siwat met representatives of KCL, UCL and Westmisnter Uni to oganise a London iGEM Meetup. Alberto, Thomas and Amrit met with Albert Fabregas, a PhD student from Guy Bart-Stan lab who is working with optogenetics. He told us that for 2D applications he can do the same with optogenetics and does not see the benefit of electricity. However, it would be cool to develop a toolkit for electronic gene expression --> we now have chemical toolkits, light, temperature, pressure and even pH toolkits to activate gene expression... but what about an electrical input?? Improvements on the wiki and human practice. Also we have 300£ in the VWR stores account, so that "we can buy cheap ethanol and get fucked up" - Josh. In the dry lab we received the carbon electrodes to test. Luis is working on a very comprehensive modelling guide. The Arduino also arrived in the lab.

Monday

In the morning we performed the colony PCR to clone SoxR from MG1655. Siwat is working on the interactive HTML Science Communication guideline. Yu is making progresses in outlining the HTML architecture of the wiki. Alberto is also working on the HTML code of the Team Cohesion questionnaire. Luis and Will talked with Tom Ouldridge, who suggested to model a square wave form of Pyo as input to GFP as output (to see resoponse of different concentration of pyocyanin). Still no response from COMSOL, is it possible that at Imperial nobody works on electrochemistry? The electrodes also have been built. Lunch break in SAF cafe. After lunch, we went to the VWR store downstairs in SAFB to pick up an Eppendorf parcel and bought some Ethanol. We did three colony PCR, the first 2 did not work (too long elongation time probably); finally the last one of the day worked.

Tuesday

In the morning we performed gel extraction of the succesful colony PCR gel from yesterday. We managed to collect 33 ng/ul of DNA of SoxR. Not a lot, but enough to engineer some genetic circuits. Also we performed the Golden Gate assembly for construct 1. We have all the parts for the 1-state system. Josh looked back at the paper and found out that even at 0-voltage, the fluorescence is marginal. General consensus is that perhaps a degradation tag on the GFP would delete the bleed that might appear. An alternative is to set the zero point at -0.5 V. Alberto, Thom and Siwat met Dr. Stephen Webster, a science communication professor, who gave hepful feedback on the science comm framework. He also really liked the philosophical perspective of PixCell (the nature-machine interface). Also today we finally received our stipends ! Also we received sponsorship for molecular Maya, a super cool software to model and animate proteins, and DNA in 3D. Also we received sponssorship from SnapGene, amazing! Next monday we have the presentation to the centre, we need to start thinking about real applications. Tom Ouldridge proposed a system to keep cells spatial separated by making that some cells survive only at certiain voltages. You can do a lot of "interesting things in non-homogenous pots".

Wednesday

Today, in wet lab the Kanamycin V3 worked but the V4 did not. In the afternoon, Thomas and Alberto miniprepped the sucessful V3. Josh updated the protocols. Luis and Will got comsol licence from a chemical engineering professor and had a talk with Tom about modelling. They decided to continue to use Matlab and maybe use comsol when it is needed. Luis and Will coninued working on modelling the redox small molecules. People were also preparing the ppt for next Monday's presentation.

Thursday

Cell growth curve on the plate reader was set up. Luis, Will and Josh spent a lot of time with Tom Ouldridge discussing about the model. Also Alberto and Josh prepared the pyocyanin solution in the fume hood, as it is very toxic. And it's an amazing prussian blue color. Ferrocyanide is a "mediterranean sun" yellow. At 5, Ben Reeve (CSO from CustoMem and former Imperial iGEMer) came into the lab to chat with us about our project and how to improve team dynamics. A flat structure is ideal. It is important to do social activities outside the lab as well.

Friday

It is Lidia's birthday today ! Also welcome to Luca, her boyfriend. The cell growth curve does not look very good. The plate reader was not shaking... that explains why perhaps... We plated agar+kan+pyo+FeCN and tested the electrode. Oxidised Pyo is blue and indeed in the proximity of an oxidising electrode there is blue spot ! this is a "tantalising result" -[Tom Ouldridge]. Will went to Westiminster for a collaboration, he had to do a voiceover for the iGEM team as he has a very deep and sexy voice.

Monday

In the morning we finished the presentation. At 12:30 presentation with CSynB. It went pretty well. Siwat's Science Communication framework was not completed and outputted (REDACTED FOR THE SAKE OF OUR AUDIENCES), in front of everyone ! Definetly an unforgettable presentation. Nice feedbacks from Danny O'Hare. Wet lab: we prepared a PCR gel, according to it colony 3 definetly has GFP but no SoxR. MATLAB is done, now it's time for COMSOL. Tomorrow we try to sort out the electrode architecture. For the team cohesion app, now it saves the inputs ad send them into an e-mail. WIKI is on its way.

Tuesday

Not a very productive day in the wet lab today to be honest. Even though Thomas performed a lot of minipreps, we are still waiting for the electrodes to work so that we can do the electrode experiment in solution. Also we need to find the MazF gene, which is a toxin for the biocontainment application. SoxR knockout should arrive tomorrow, so we need to do the comptenent cell prep again. Alberto finally went to the Italian Consulate to renew the passport. Lidia, Josh and Yutong spoke with Marko regarding the promoter library construction and BASIC assembly. For the Oxford collab, we talked to Marko from Baldwin's lab. He's helping us, he gives us the sequences for the promoter architecture. We've blasted pSoxS to find other promoters from other organisms. We design the promoters, send them to Oxford and then we characterise them with Voltage and they with NO. We need to start thinking about what submitting as best new part (MazF ?), composite part (maybe the promoter architecture ?!) and the improved part (SoxR?). The dry lab team got a bunch of holes drilled in the electrode and they fit quite nicely. A concern is that the current through all the electrodes has to be same for the potentiostat. At the moment what is stopping us to set up a solution to Pyo/FeCN is just a power supply. Also we are using Robert Bradley's potentiostat, but he needs it... so maybe we should ask for someone to sponsor us a potentiostat. After lunch, Siwat, Alberto and Thomas went to the science museum to do some linguistic surveys... A lot of people do not know the term "Synthetic Biology" and almost everyone confused plasmid with plasma. What about doing something this friday as a team after work...the iGEM social! Options are: pub, dinner or park. We decided for a custom made quiz day in the park on friday at 5 (the geekiest choice). We are at Imperial after all... Also the outreach team at Imperial replied and they are on board.

Wednesday

Today we could not get into the lab at 9 because we got told off by the lab technicians... for reasons still unknown. We worked a bit in the lecture theatre. The lawn of cells grew on the agar plate with Pyocyanyn and Ferrocyanide inside. They grew fine. Also we penetrated with eletrodes but did not observe any GFP expression unfortunately. Maybe we still need to wait for it to be expressed. Josh, Alberto and Luis perfomed the electrode in solution experiment. Below it's a picture of the DIY set-up: hopefully we have some practical engineers in the team ! We measured the oxidation state of Pyo and FeCN using the molecules absorption peaks over time, measuring with the Plate Reader. The results have not been analyse yet, but they look quite random (read stochastic). PCR to check construct 1 still didn't work. I wonder why can't we just sequence it? We'll have to do it anyway, right? Parts for construct 2 have been designed. Also picked TetR and RFP parts from the iGEM Distribution Kit 2018.

Thursday

In the morning, we checked the plate reader results, but no fluorescence was detected. BAD NEWS: DJ1901 IS KAN RESISTANT: problem is that also the construct is Kan resistant. We did PCR, 6 of the 8 Amp promoters fro Construct 2. transformation for the biobricks did not work from last night: we did not know if it was from bad competent cells. We therefore performed another Assembly with an Ampicillin resistant backbone. Also we did not see any fluorescence in our agar plate incubated with C1 and redox molecules. We know that the voltage is the right one, but are we sure the solution is oxidising?

Friday

First thing in the morning we checked the plate reder. No fluorescence and no cell growth either... The blank showed increase in Absorbance (?!) so presumably we observed a contamination. We did a lot of gel extraction today (really a lot)... all of them worked fine. Also we prepared comptent cells agains. Very busy day in the lab. Now we have all the parts in the freezer for construct 2. Also we received a transilluminator as gift from the Teaching Lab. Fortunately most of the PCRs today worked. The dry lab has been looking for components for more detailed design. And it seems that it's possible to have most of the components from Nexperia, which makes sense as it has the monopoly of transistors! T.O. suggests that the modelling could be the patterining of pyocyanin as a function of potential. Also the stuff about the device screening is good. Actually there's plenty of material for modelling. At 5:30 meeting with T.O. and then off to the union for the quiz night. PixCell Social: QUIZ NIGHT After several rounds of gel extractions, we managed to leave for the Union. We were supposed to have the quiz in Hyde Park. However we ended up at the union because of the rain. Rounds: Historical figures - Tom and Thomas Paintings at the National Gallery - Diellza and Alberto Japanese culture - Siwat and Shivang Random and controversial facts about singers -Ismael and Yutong Legumes or swearwords ? - Josh and Luis Cheeses - Lidia and Luca References in PIXAR movies - Will

Monday

In the morning we started drilling some holes in other 6 eppendorf tubes to fill in other electrodes and redo the electrode experiment in solution. This time with improved sterility techniques !! and just pyocyanin as redox molecule (no ferricyanide). Cells with Construct 1A inside (hopefully) have been suspended in the modified eppendorfs in a Amp + LB + Pyo solution. Positive controls with constitive GFP have also been suspended. To test the electrode set up we are oxidising /reducing a pyocyanin solution. Results tomorrow. Josh is ill. Rodrigo is back !

Tuesday

"Eureka" day! First thing in the morning we checked the tubes stimulated overnight and there was aesthetically-pleasing GFP expression in C1A (qualitative result). Surprisingly, the "control" with GFP constituively expressed does not have a significant fluorescence. Also there is no difference between stimulation with oxidising potential and negative potential....might be that we stimulated it overnight, and not for 20 min as in the paper. Perhaps a redox coping mechanism counterbalances the oxidative force at the electrodes? Lunch in farmer's market. Webinar with Promega. Meeting at 5 with Rodrigo and then setting up a growth curve with C1A and controls for tomorrow's electrode in solution experiment, for more quantitative results. Dry lab: It's important to prove that we can oxidise and reduce the solution. Sofar we still havend demonstrated it with color changes... we managed to do it however in agar... so perhaps diffusion is a problem ? It's time to escape from equilibrium.

Wednesday

No one is in the lab until 11, so we cannot start with the experiments. As soon as the lab doors opened, we checked the growth curve experiment, which are not summarised here but they are interesting. Also we observed the plate containing C1A (bottom plate) and it is fluorescent. However, there is no patterning (yet). Josh is still sick. We performed other PCRs and gel extraction of C1A + degradation tags. We are meant to do Golden Gate but it's postponed for tomorrow. We prepared the sequencing primers for C2. Lidia and Diellza went to VWR store to do some lab stuff shopping. Also we prepared more LB and water. In the dry lab we thought about doing another electrode set up with the salt bridge. We had a group dynamics problem in the team between Yutong and Shivang. This was brought up during the 5 pm meeting and we discussed all together as a group. Hopefully it is solved now, or at least exposed to the group attention.

Thursday

In the moning we set up a new electrode stimulation experiment with both Pyo and FeCN. We also performed Golden Gate Assembly for the next constructs. We collected results from the plate reader experiment number 2 and they look good again (they weere replicates from yesterday and we got the same output). Tonight we are setting up a new plate reader experiment (a plate reader a day keeps the doctor away). After lunch we met with Francesca Ceroni and she was amazing to be honest. She is thinking about applications of PixCell in mammalian cells and said that probably the leakiness of our system could be due to the fact that our construct is plasmid based... perhaps it would be interesting to integrate it in the genome, so there is only one copy. Josh is still sick. Siwat is back and Shivang is sick.

Friday

Josh is finally back. Alberto set up a new growth curve with different Pyocyanin concentrations. The plates we incubated yesterday with redox molecules and electronic stimulation did not work, as we put the wrong antibiotics. Alberto, Siwat and Thomas met to work on the Human Practice Gide draft. From collaborations we hear back from Joe Windo to arrange the New Scientist Live booth. Luis and Will tested the agar salt bridge but no good results. Also we ordered silver electrodes, which hopefully should work faster! We still don't have vouchers for sequencing. What is happening with Source sequencing ? Meeting at 5 with Tom and Rodrigo.

Monday

In the morning we organised what to do in the lab for the next week. Not a productive day in the wet lab... Alberto is setting up a new plate reader experiment using DJ901 cells, with C1A construct inside, C1A + ssRA tag and control. hopefully tomorrow we have good results. Lab meeting at 5pm.

Tuesday

As soon as the doors of lab were opened by the guardian Ismael, we started to prepare new plate solutions and constructs. Golden Gate is still not working. Hopefully Diellza does not give up and she is trying again and again. At 12 Prof. Danny O'Hare came into our lab and spoke his words of wisdom. Apparently, oxygen at the interface of carbon electrodes reacts and forms hydrogen peroxide, which interferes with our oxidation signal (pyocyanin). He suggested (as already Tom and Thomas thought) that we could add some reducing molecules in the solution. Basically the idea is to reduce chemically the solution as a whole and then oxidise locally with the electrodes. Possible reducing molecules we can use are ascorbate, DTT and even reduced ferrocyaninde. Also sulfites can get rid of the oxygen, to prevent H2O2 formation. Also it is important that we calculate the molar absorbitivity of pyocyanin to make sure we can detect its oxidation with the plate reader. Affter lunch we started testing Dani ideas. We prepared solution with NaSO4 and reduced ferrocyanide to make new plate reader experiments and plate sert-ups. Set up a new growth curve (a plate reader a day keeps the doctor away...) Also we sent the e-mail to Tanya (the first author of the paper we are using). Lab Meeting with Rodrigo at 5.

Wednesday

We received the sequencing results back. We have construct 1, with ssRA tags confirmed. We saw the results from the plates ffrom yesterday and they look promising. We don't have background fluorescence in the presence of reduced ferrocyanide in the agar plate. Overnight plate exeriment and solution experiment suggests that sulfite does reduce GFP fluorescence but cells in the plate were able to recover as they have interface with oxigen.The plates contained also sodium sulfites, that gets rid of oxygen (which mimics the reducing portential of the electrode). Now it's time to do the same, but with the elctrodes. We are going to do it today. Hopefully by the end of the week, we have some solid results. Golden Gate did not work again. The positive contro also did not work. This suggests that there is something wrong in the golden gate mixture. Sue, the lab technician, reminded to Amrit that we need to clean more. Everytime we use something, just clean it afterwards and tidy up the lab every night. So we are getting back the cleaning rota. And also let's come to the lab earlier, so we don't have to leave at 22 every night... After yesterday's meeting with Danny, we thought it would be fair to acknowlede him and Rob Bradley as "Special Advisers" somewhere in the wiki. As they helped us a lot. Siwat came up with a cool idea for a PixCell-inspired board game. Each character has a bacterium and you can choose which parts to have. Every round you try to conquer a tile and you have a set replication rate. We plan to use it as an education tool for our outreach events. Josh is writing outlines for the wet lab section of the wiki. Yutong is now asking for content in the wiki. Please give her content. The bioengineers spent most of the day doing some heavy maths from Danny's paper. Apparently, the maths is not as easy as Tom Ouldridge said yesterday. Lab meeting at 5 pm.

Thursday

We have fluorescence and we can modulate it using NaSO4 as reducing agent to keep the system off. But as for now, still no spatially-defined patterns... The bioengineers have done a lot of maths (the one that Danny sent us). And it makes sense and is accurate enough to study our system. Essentially, the results show the concentration of redox molecules as a function of the length from the center of the electrode, and it is also normalised for the radius of the electrode. In the wet lab, there was a shift: Diellza is doing the electrochemistry experiments and Josh is trying to make the Golden Gate work. For Alberto is plate reader time as usual. We need to find a good positive control for the experiments. We also got our hands on iLOV (GFP-like that fluoresces in anaerobic conditions) from a PhD student from Karen Polizzi's lab. Sequencing results came back, we are missing an A in the degradation tag and this could cause a frameshift. We don't know yet if it's the polymerase that slipped or its' the sequencing. If it's the polyerase, this could explain why we only have a 2-fold decrease in fluorescence rather than a 16-fold, as predicted by the modelling. Lidia sent the order for the promoter library to IDT. We did cyclic voltammetry and square wave voltametry for different solution at varying concentrations of Na2SO3 (+Pyo +FeCN + NaCl). Using the result we are trying to determine the optimal voltage to apply onto the agar plates. Siwat and Ismael designed the board game. It sounds like a lot of fun. Lab meeting at 5 without Tom nor Rodrigo.

Friday

Alberto is sick, so Yutong will take over the journal. We did another GG, becuase Nico's (apparent GG expert) GG didnt work. We proved that we need FCN(R) in the plate for it to work. At 2pm (UK time) we Skyped with Tanya Tschirhart, the first author of the Nature Communications paper we are basing our work on (she is super nice). We tested higher conc. of Pyo and used 2 separate chambers for oxidising and reducing, as Tanya suggested. We were able to get the oxidation and reduction peaks AND beutiful cyclic voltamitry curves. She also suggested that we should try to use grow cells in the plates as a way to get rid of the oxygen, we can try later. Sadly, PCR did not work again for RFP TetR so we need to redo it. (T_T)

Monday

No Bank Holiday does for us. Union night. New logo idea from Virginia:

Tuesday

Alberto is stil sick. Thomas flight from Austria has been delayed. GG did not work, again...

Wednesday

Today we arrived quite late in the lab to be honest. Alberto and Thomas are back. Josh and Alberto designed a new plate reader layout and experiements to characterise the constructs. Lidia met Marko to discuss about the BASIC protocols and designed protocols for the promoter library characterization. Perhaps we can use the DNA Foundry to combinatorially characterise the library, very automated and high-throughput! At 5 p.m. meeting with Tom. T.O. on the plate reader results: "Good evidence that sulfite is causing reduction of pyocyanin in solution, if you have enough of it". But what is it then that causes the ring of death around the electrodes ? Also it's time to start assembling the biocontainment construct. Regardind the agar experiments, we need a way to quantify the oxidation/reduction level of Pyo locally around the electrodes. Can we measure the absorbance of a plate directly ? Josh argues the there would be too many refractive interfaces messing up with the measurements.

Thursday

Wet Lab: We have the results from the spotting plate experiments. The cyclic voltammetry showed a peak for sulfite. Therefore it is likely that the bacteria are implicated in the redox process. Josh argues that "this is a ferrocyanide-driven, pyocyanin-mediated redox reaction". The amplification of the iLOV gene is not working. Amplification of the backbone is working at specific temperatures but it varies everytime we run it. We need to try it again tomorrow. Alberto set-up the plate reader experiment measuring oxidation of Pyo over time, in solution with FCN(R) and sodium sulfite. Dry Lab: Danny gave us some gold electrodes. The modeling guys finished the PCB circuit. Also we can use AgCl to link the PCB to the circuit. Also a lot more maths and a lot more of learning maths to model the electrochemistry. Human Practices: We could not meet Prof. Sternberg. We designed a new survey more focused on the ethics for tomorrow. At 5pm meeting with Rodrigo.

Friday

A relatively sunny day. Wet Lab: The plate reader experiment from yesterday (Oxidation of Pyo over time, in solution with FCN(R) and sodium sulfite) shows no effect of ferrocyanide on pycyanin. For the Oxford collaboration, we received the gblocks and we are going to miniprep them next week. In the meantime, Joe Windo received them a couple of days ago. Why ? Alberto made a new spotting plate but this time with Pyocyanin. Diellza ran the PCR to add the ssRA tag to C1A. Tomorrow we know if it works. Shivang did some PCRs for the Valencia collaboration but they did not work. Josh, Diellza and Alberto prepared new redox molecules solutions, to completely replicate the electrochemistry of the Nature paper. Human Practices: Thomas and Alberto went to the Science Museum to survey people. The hypothesis is that there is a correlation between background knowledge on biotechnology and its acceptance. Tom suggests that we should change the survey to be more focused and integrated into PixCell. Siwat incorporated all the feedbacks from KUS iGEM team into the science communication framework. He is also contacting the Debate Society and looking for speakers. Wiki: Yutong is trying to finish the Lab Overview and Attribution Page. Dry Lab: schematic of the PCB is finished, we just need to check with Paschal. We have decided to change the TX/TL model from SoxR dbeing dependedn of the percentage of oxidised of Pyocyanin to SOxR being dependend on the concentration of Pyocyanin in whatever state and assuming full oxidisation. Luis made agar salt bridges. At 5 pm meeting with Tom.

Saturday

"Chill" saturday in the lab. Set up a gel plate reader experiment. Lidia set up a new spotting plate experiment.

Monday

Yutong is doing the attribution page, with pictures of Dani and Francesca Ceroni. Also she is making a "For Judges" page, with a checklist demonstrating how we meet the medal criteria. Josh, Siwat, Yutong, Thomas and Alberto went to the Science Museum to survey more people. Perhaps the survey is a bit hard. We are designing a new survey now, more specific about PixCell and quicker. Sternberg still did not reply. Meeting with Tom at 5pm. Team Cohesion app is almost ready. Will chlorited the silver electrode to make it more stable. Also Luis did the curve fitting. They need more data. One suggestion from the PhD is to do the plate experiments, to make the bacteria in the agar. Tom says: "How do we present this in a way in which people can understand (for thursday withGuy and karen and Kirsten)? Leave all the applications to the end. Not as we would present at the Jamboree. What are the key challenges ? Getting the electrochemistry to work. getting to work on the plate, getting the cell to respond to the signal, getting the cell to work an a plate. Give them a very clear this is the framework opf the project. Without giving too much details on why it is so exciting. Here is how we are trying to reach that medal and this is how we got so far "

Tuesday

Ismael presented to us what he is going to present at the BioMedEng Conference on Thursday. New plate reader experiment measuring at OD380 + OD390 + OD400 to investigate if pyocyanin reacts with sodium sulfite to produce pyocyanine sulfonate (absorbs at 400 nm). Will plotted the results of the GEL plate reader experiment in 3D using MATLAB. Very aesthetically pleasing results... but do they make sense ? We will find it out in the next plate reader experiments Meeting with Rodrigo at 5 pm. Quote of the day: "That's quite finnicky"- Will talking about a MATLAB function Siwat, Thom and Alberto had a Human Practices "meeting" at the Union... Josh and Ismael joined them for dinner Streaking plates at 23 pm after the union...not a great idea as we put the cells in the wrong plates!

Wednesday

We arrived quite late this morning. Alberto has red eyes. Siwat arrived late...pehaps he could not handle yesterday's beer... Will and shiv are not in. We received sequencing results and new primers from IDT. Yutong made a lot of agar plates with different concentrations of redox molecules. Lidia prepared a new spotting plate experiment to measure the time of activation of our circuit. Alberto did not perform a new plate reader experiment. Luis plotted the plate reader data on MATLAB

Thursday

Good results from the plate experiments, we found a new condition (Na2SO3 concentration) where reduced Ferrocyanide turns off the system, and oxidised Ferricyanide turns it on. This is the sweet 0.025 % spot. We sneaked in the BiomedEng conference to get free lunch. Alberto ate more Kinder Buenos We aldo listened to Ismael and Rodrigo's Presentation at BioMedEng18. Yarrowa Lipolitica is very "trendy" - [Rodrigo]. At 15 we received Karen Polizzi, Guy Bart-Stan and Tom Ellis in the lab, Rodrigo and Thom were there also. It was a very intense but constructive feedback. Tom Ellis threw a brainbow ball at Tom Ouldridge. He is quite a character but a furnace of ideas. Karen is always positive and has always the right comment to say/point out, it's amazing. Guy-Bart was calm and rational. We kind of sorted out which parts to assign to each medal.

Friday

Very busy day in the lab. Josh, Yutong and Siwat prepared the plate stimulation experiment. Alberto filled up a 96-well plate for the C1A_1 experiment. Lidia and Yutong went to talk with David McClymont, head of the DNA Foundry. They are excited about the project and we can use it. Let's bring automation to iGEM. Why wasting hours pipetting when you can use that time to design cooler experiments? We also had the lab safety induction in Bessemer, in order to use the lab next week. It is a very well equipped lab. At the end, the transformation for C1A-ssRA tag did not work. As a result, Alberto could not inoculate the microplate. Kirsten Jensen and Paul Freemont's student came into our lab for some feedbacks after lunch. In particular, we discussed whether it is fair to use the Foundry as part of an iGEM project. It is true that not all other universities have access to such technological wonderlands as we do. However, we want to use it to make and characterise new parts to send to the registry and share them with those who don't have access to foundries, so isn't it a win-win for everyone ? Lidia, Yutong and Josh then went to meet Marko. Marko is always nice and helps us so much. Let's also bring BASIC to iGEM. We are so generous... Luis drilled 120 holes in a total of 28 plates. "Will cut a shit-ton (30) of electrodes. Luis polished them. Will also chlorited a shit-ton (12...) of silver wires. It was a beautiful amplification of working efficiency" [Luis]. Will is also thinking of opening a silver chloriding company called "NaCl bois". Unfortunately, they now have to glue all these electrodes into the holes. Luis did grocecry shopping on Ocado, while chatting with Josh. Luis finally ordered BreakOut boards (15), which costed 140£. Also there are bad news: Sigma Aldrich (now Merck apparently...) made a mistake with our order and there is no more Pyocyanin until next friday. We only have 5 mL left and we are rationising it for the most significant experiments. Therefore , we could not do the whole plate stimulation experiment, which also took longer than expected. Tom Ouldridge came into the lab at 5 pm and we took a picture for the safety form (below) Today is the last lab meeting for Diellza...

Saturday

Routine lab day

Saturday

Josh, Diellza and Lidia have been working in the lab. Transformation and Oxford Collaboration/promoter library. MazF, Gp2 and MelA have been ordered as gBlocks for the "Application" parts. We wil do the voltammetry with copper and tyrosine for MelA. Hoping that the copper does not interfere with our electrochemistry. The "Let's talk about This App" is officially out (headquarter at Shiv's place, where the Arduino is) .

Monday

Lunch in SAF. Alberto and Yutong went to the VWR store and bought a new primer freezer box and a super cool beehive Falcon-tube holder. Best investment ever. Alberto prepared a new C1A_1 96-well plate and froze it for future use. Quote of the day from Tom Ouldridge to Will : "your maths is very impressive, for a person that has received less mathematical training than I have". Nothing new in the modelling front, "it has been like hitting the head against a hard wall of maths today" - [Will] Rodrigo came into the lab after lunch. Let's start writing a document with all the medal criteria and how we tick the boxes. T7 pol has been ordered as gBlocks. We finally received new Pyocyanin. The transfromation for the ssRA tag still did not work. If it does not work again we will just ignore that extra strain. Alberto is preparing a new sodium sulfite plate to test reduction kinetics in agar and in solution. Perfect timing for the team cohesion app, which has been used today for the first time, after one day after being released. Shiv and Lidia had an argument regarding the Outreach poster. This journal remains neutral, therefore no other pixels will be wasted on this issue. But let's talk about this! Regarding the survey for Human Practices, we need to think about which scientific method to use to analyse the surveys. Chi-squared? Possible questions: What is your view of using GMOs for food? Favourable Unfavourable, Not sure What is yor view of using GMOs to produce anticancer drugs? Favourable Unfavourable, Not sure What is your view of using electricity with cells for food ? Some fun stuff: We need to pick a color for the PixCell sweatshirt. Yutong and Alberto went to Bessemer with Amrit to perform Heat Shock Transformation. Cells were incubated for 1 hour in the Phillip Lab and were plated at 22.

Tuesday

The transformation plates show small colonies. We are growing them with Pyo, if they grow and glow it means they worked ! Hopefully they do it by tonight, so Alberto can set up the C1A_1 plate reader experiment. Alberto and Siwat set up a new gel + solution plate reader experiment to measure oxidation kinetics of pyocyanin in the presence of sodium sulfite. They are using the plate reader in Ouldridge's Lab, which has a fancy injector that saves us 1 hour of manual work at least and allows us to measure recution of pyocyanin in real time. Josh created a collaborative playlist on Spotify and we are going to launch it as the iGEM Radio. Each tem member has the right to add max 10 songs. For Siwat it's either Rasputin or Yeyeyey What's Going On. Country Road does not count. Lunch in Farmer's Market. Topics of discussion at lunch: disney remixes, stochaticity of living systems, anarco syndicalist utopia and other weird stuff as usual. Dry lab: Curve fitting done using global algorithms Bad News: IDT e-mailed us saying theat the gBlocks fot TerR-RFP (C2A) does not work. What do we do now ? Other bad news: we ran out of sequencing credit. The stress is bulding up in the team. Diellza left already. Josh is leaving in 10 days and Lidia as well. Yutong will take Lidia's role, Alberto will take Josh's role. Also who is presenting at the Jamboree ? Will, Siwat and Lidia ? Also, who is going to the New Scientist Live ? The Easter egg PixCell game fro the Wiki is done (in HTML format). Thank you Luca!!!! Josh, Alberto, Thomas, Siwat and Yutong had dinner at the union. At 23 they then returned to the lab to finish the plate reader experiment.

Wednesday

The plate reader experienced contamination. Alberto will do it again today. Yutong made plates for the electrode stimulation. Alberto went to Bessemer to pick up the plate from the CLARIOstar. Results are bad. What a shitty day today (result-wise). But it's fine we cannot have good results every day. At the end frustration in the lanb pushes to perform more clever experiments. "Diamonds are made under pressure, if there is no pressure you remain carbon. And we don't want to remain carbon, do we?" [Alberto] We measured OD390 (abs peak of pro) in LB-agar and in LB (without cells) to measure oxidation profile over time. The results are hard to interpret... (reduction of pyocyanin by sodium sulfite over time) ? Can some electrochemist explain what happens after 6 hours ? Is this the proof that we are generating pyocyanin sulfonate? We sent the promoter library to sequencing. Diellza made primers for assembling C1A in storage vector. Josh is stimulating plates with electrodes. Surveys at the Science Museum. Siwat met some Thai people. People seem to be surprisingly in favour of GMOs. Will has been working on explaining all the maths and physics of the model in a very comprehensive LaTex guide. It's just doing the justification for modelling: concentration profile distributon with the flux and all the full derivations. Luis discussed with Pascal but we did not get much from it. Luis also glued some electrodes. (REDACTED) arrived at 18 in the Phillip lab. I am afraid I cannot specify here who I am referring to. Yutong bought a Winney the Pooh peluche as a rebellion against the CCP. Also, today we started to move out from the Phillips Lab. Alberto and Josh got kicked out from the lab as they stayed past midnight.

Thursday

Despite loading the 96-well plate after a couple of beers and at midnight, the results are "tantalising". GFP/OD600 values for C1A at different [Pyocyanin] are as expected. Yutong prepared cell cultures for the plate reader experiment and for the promoter library. Lidia worked on the promoter library. Alberto went to Bessemer to pick up the new plate from the CLARIOstar and performed the second day of the C1A_1 plate reader experiment. Siwat worked on Human Practices (nothing of value was done this day). Other stuff in the Lab: We did not get the iLOV and RFP for the library characterization unfortunately. Luis discussed with Paschal about an important change in the power of the PCB (too technical to be mentioned here). We had lunch in Queen's Lawn, as it was a relatively sunny day. Luis was modeling alone today, as Will was not here unfortunately. Josh outlined the Abstract for the wiki. We moved out stuff from RCS to Bessemer. We had some beef with the lab technician, as she demanded to pay back the nanograms of reagents we used. But wasn't the department of Life Sciences supposed to sponsor us ? It is sad how vile money interferes with the science. Yutong, Lidia, Thomas and Alberto went out surveying at the Science Musuem and Natural History. Rodrigo arrived in the lab at 5 pm. Meeting of the day as usual. Apparently, Yutong has a phobia of walking on things where you can see through.

Friday

Very busy day. We officially moved from the Phillip Lab to Rodrigo's and Tom's benches in the labs in Bessemer (Centre for Synthetic Biology). We spent the morning and the first half of the afternoon tidyng up the lab and packing all the equipment. The lab felt incredibly empty. Lidia talked a lot with Sue. We transferred LB solutions from the Duran bottles belonging to the Teaching Lab to some Falcon tubes. Lunch in the Junior Common Room. Surprisingly, Luis did not prepare his lunch today. After lunch, we went to the Phillips lab for the last time and to receive the final blessing from Sue. We moved the Plate Reader with a trolley. We did several trips to move all the stuff. Bessemer has been officially invaded. Once in the new lab, we unpacked everything and tried to fit everything in the tiny space we have available. We have: Rodrigo's master student bench (who is on holiday until October), the space on the left to Javier's bench, Ismael's bench (not all of it) and perhaps Amrit's bench, but he did not look very keen about this. At 6 pm, we had the team meeting in the Entreprise Lab (where there is free coffee and beer.) We should do meetings there more often. We launched the new lab by staying late again. After the meeting, we went back as we had to perform some plate experiments. It was again a race against against time, as at midnight all the automatic doors automatically close. In order to speed up the protocols, we let the Petri dishes dry in the freezer. It worked. We seeded some cells on those plate and ran outside. Josh, his girlfriend, Lidia, Alberto and Yutong tried to go to the Union, but it was too late and therefore the bar closed.

Saturday

Lidia and Yutong worked in Sir Alexander Fleming (SAF) building to perform some BASIC assembly for the promoter library. Siwat was worked in SAF as well, on the PixCell board game. Josh, Thomas, Diellza, Alberto, Luis and Shiv worked at the interface between dry and wet lab in Bessemer. A new plate reader experiment was set up, using phenazine methanosulfonate (PMS) as an electrochemical mediator altervative to pyocyanin. If we can oxidisie it and reduce it using our set-up, it is a very good result (as it is cheaper and way more effective at lower concentrations). We had dinner all together at the Oriental Canteen near South Kensington station. with Henry (Josh's friend who was in the iGEM winning team 2016, Ecolibrium). As he is a board gamer, we had a chat about potential hack strategies regarding our board games. We went to the Union after. Topics of conversation at the pub: DNA encryption, Tinder as a new platform for communication, stochastic vs deterministic living systems. Everything normal as usual...

Sunday

Pyocyanin, Pyo + FCN electrochemistry. We pulsed plates overnight. Josh and Alberto set up a plate reder experiment with Phenazine as an alrternative to Pyocyanin.

Monday

A relatively sunny day. Glorious results from the plate reader with phenazine. We have the same GFP output than Pyocyanin bt with 1/100 of concentration and it's 40x cheaper (and non toxic). This is a promising result for our Integrated Human Practices (and the project in general). Alberto and Siwat stayed in Bessemer late loading plates.

Tuesday

We are doing more electrode experiments. We are also doing a lot of plate readers.

Wednesday

Lunch in Farmer's Market Meeting in 4th floor Bessemer with Rodrigo. Presentation to decide who is going to present at the Jamboree. Board Game will be printed tomorrow. Shiv's poster for the New Scientist Live is almost completed.

Thursday

In the lab there are a lot of bad news: LB is contaminated, the plate reader Ferro_1 experiment had contamination in the blans , Josh's assembly did not work and we forgot to autoclave water... We said this to Albert, the PhD student in Guy's Lab and he said: "sounds like a normal day in the lab". Amrit is finally back. Some good news: We received the Benchling t-shirts ! Also the modelers have assembled the arduino with and they can control +ve and -ve voltage in the electrode array.

Friday

Shiv and Ismael are at the New Scientist Live with Oxford! Yutong, Thomas and Lidia had a moving day, which was a pain. Bessemer therefore was quite empty.

Saturday

Siwat doing plate reader Thomas doing electrode stimulation Lidia and yutong are in SAF doing the promoter library Shiv is at the New Scientist Live with Isma. Alice and Rodrigo passed by our stand the fair.

Sunday

Siwat is doing more plate reader Shiv is at the New Scientist Live, Lidia is here as well.

Monday

A freezing day. In the morning we checked the results of the plate reader with pyocyanin. We picked a new concentration of pyocyanin = 2.5 uM, when cells grow healthy and express a wonderful amount of GFP. We had lunch in JCR. Shazia came to take back the Eppendorf equipment on loan. Good news (finally...): it looks like we have spatially patterned GFP !! Now let's not say cat before we have it in the bag: Thomas is redoinfg the same exact experiment to check if it is luck or out system actual works. We had a meeting afetr lunch with Rodrigo and Thomas to summarise what we have done and make a plan for the coming weeks, which look very intense. We decided the overall theme of the preseantation and assigned roles. Josh stayed late in SAF to do Assembly of the application plasmids. Yutong, Thomas and Alberto stayed in Bessemer to do wiki, redo the electrode stimulation and sulfite plates experiment. Unfortunately Alberto could not finish the experiment as we got kicked out by the security at 12 am.

Tuesday

Another freezing day. Lunch in Farmer's Market. Today is Thomas's Birthday. As a present, we have a proof of concept! The same experiment which showed spatially patterned GFP worked again. We showed the plates to Nicolas, Alice and Albert, who looked surprisingly satisfied with the results. Yutong and Alberto are working on the banner. Lidia is working hard on the library characterization. We have no news on the dry lab guy. This working situation is a bit dispersive. Luis declared that he feels a bit alone. Let's talk about this! We finalised a date for the debate for human practices with SynBIC: it's the 9th of October, after Tom Ellis preseantation!

Wednesday

Today is Josh's Last day... we are going to miss him but we wish him good luck in Cambridge! Lidia and Yutong worked mainly in SAFB on the promoter library. Alberto and Lidia then began the library characterization with the plate reader. Alberto used a multichannel pipette for the first time, amazing! Thomas redid the stimulation experiment and worked again. We now have big hopes for when the PCB arrives. We had a big meeting with Tom and Rodrigo at 5 pm. We revised who is doing what in the next weeks and revised the parts we are going to submit. Also we focused more on the presentation.

Thursday

The promoter libray results from the plate reader looked interesting. More info are going to be on the wiki. Ismael brought Ben's cookies for everyone, thank you guardian of the welfare! From the results os the agar plate experiment, we finally picked the right (lowest) sulfite concentration to turn the system off= 0.02%. Our plates look like galaxies! Shiv is working on the banner. Siwat is working on the presenation. No news from the bioengineers...apart that Luis had bread and hummus for lunch.

Friday

We collected the results from the "ferri" plate reader results. Unfortunately the plate stopped working at 1 am. So Alberto has to do it again today... At 12:30, presentation with the centre of synthetic biology. Questions that came up: Wooli: IN the introduction part put some iterative sentences, we are jumping in the project too fast. Rochelle: do a better job at explaining that you are trying to achieve. First sentence should be: we are pixcell and we are trying to....You need to tell a story. Also make it more accessible. For example, PCB what is it. Start with circuitry, not application. Ismael: try not to split information from the same section into different ones Nicolas: put log scale and make prettier data. Also what information from modelling is actually flowing in the project? Michael: there is no clear application of what our system can do better than optogenetics. It is not application-driven Alice: it would be good to mention that we can explore not only electronics- > biology, but also biology--> electronics. This bidirectionality of communication is one of the main advantages of electrogenetics! Karen: I did not know where I was in the nabar at the top Albert: in terms of experimantal part, it would be nice to have each slide to have a title that summarises the result, e.g. "We proved that pyocyanin turns on our system"... etc.. Have it more compartmentlised so that people can follow the story also of experiments. Kirsten: don't be afraid to change your presentation. Also best results should be on the wiki. Lunch in JCR. Today is the last day for Lidia. We have some issues with the Oxford people for the collaboration. We stayed in the lab to complete plate reader and basic assemblies.

Saturday

Lab Day

Sunday

Intense Lab Day

Monday

Today is the official first day of term. Imperial is crowded again. Also it is the first day of Yutong, Siwat, Thomas and Alberto alone in the lab. The application constructs seem to have worked. Yutong is picking colonies to characterise them. Alberto has completed the data collection for C1A characterization. Now we can start to characterise the promoter library. We have a sexy sygmoid! Final preparations for tomorrow's fresher fair. Also there is a new student in the Ouldridge's lab,and he did BioMod!.

Tuesday

Today we were at the Fresher's Fair of Imperial College, resenting our poster at the Synthetic Biology stand. For the bioengineers, it's lecture time. Siwat tried to do the PCR for the silver medal. There was a lot of PCR spam on the whatsapp group

Wednesday

For the life scientists it's lecture time as well. It's going to be a very intense month. Yutong is testing the application constructs. Alberto is finalising the plate reader experiments. And plate the cells in solid cultures with the final conditions. Thomas is pulsing the plates waiting for the electrode array. Siwat is listening to Structural Biology lectures and then he's trying the PCR again. Shiv helped him on the PCR and finally we have the band as expected. We wouldn't have ticked the silver medal criteria without that band... Yutong, Thomas and Alberto went to sports night.

Thursday

Good results from the agar plates with the final conditions. Everything fluoresces as expected. The second years are going to lectures now. Interesting lecture by Prof. Mark Isalan on Directed Evolution and Turing Pattern. I am glad you asked...

Friday

Today there are good news: the Gp2 construct in oxidising conditions seems to grow way worse than in reducing conditions (and than the controls). Let's send it for sequencing for the final proof that it worked then. What a glorious day. Celebration at the union with Thomas, Yutong, Alberto and Ismael.

Saturday

Diellza is back. Since we ticked all the medal checkboxes, construct 2 is now officially under construction. Construt 2 assembly failed.

Sunday

Let's try to reassemble contruct 2. In the wet lab, Yutong is working on MelA and wiki. Alberto is in the lab characterising Gp2 and MelA plate reader. The final plate reader experiment for the library characterisation is done. Thomas is fine tuning the electrode stimulation experiment. Lidia, Josh and Diellza are woking on the judging form, wet-lab wiki and protocols respectively. The bioengineers are analysing data and producing pretty figures.

Monday

Big meeting with everyone . Lidia, Josh and Diellza joinied in via skype. The survivors on campus met in Tom Ouldridge's office. Rodrido was there as well. This week is going to be intense. Many important deadlines ahead. Also there is an issue with the part library: A1 construct did not show fluorescence in the plate reader experiment. This means that at the moment we don't have a sygmoid onto where to map the endpoit data of the other 48 constructs. This exeriment has to be urgently redone and it is now a high priority issue. Also in the priorirty list is creating a parts page on the registry and sending DNA to Boston

Tuesday

The wet lab team worked in Bessemer on plate reader and electrode fine tuning. At 6 pm we had iGEM Presentation with SynBIC and the Socio-Ethical Dialogue. We talked after Tom Ellis introduction to synthetic bioloy and John Collins on entrepreneurship. Approximately 80 undergatuates student were present. “E. coli is the ultimate capitalist. It basically evolved for fast growth and competition, not stability” [Tom Ellis]. Also Siwat memorably asked in front of the audience “does E. coli have morality” ? We still don’t have the answer to this question, but we are working on it. Feedback:

Wednesday

Everybody is working on the judging form. We received the PCB. Will and Luis started to assemble it. Unfortunately some critical components are arriving on Thursday. So we don’t think we can present it on the wiki. Perhaps we can do the big show at the presentation ? Alberto set up the pyo plate for the library.

Thursday

We have a sigmoid for the library characterisation. Everybody is working hard on the wiki. These are stressful times, but remember that PixCell relies on oxidative stress.

Friday

A relatively sunny day. Judging form freeze. The caffeine concentration has increased exponentially. Yutong had three espressos in the morning, which to be honest is nothing by Alberto’s standards. Everyone worked the judging form. It was impressive how much stuff a team can accomplish in so not enough time. Also should we get some more data for the library characterisation ? Diellza came back to Imperial ! We had a urgent meeting with Rodrigo and showed him the heat map with the fold induction. We have orthogonality! Also the PixCell sweatshirt and t-shirts arrived. At 8 pm we had more coffee in the Bioeng staff room. Alberto and Shiv set up a new plate reader to mine more data for the library. They had to hide in the darkroom in Baldwin’s lab to stay in the lab later than midnight. Everybody reveiwd the chapters for the judging form on a collaborative google doc. Everybody review all the parts and added their inputs. Such a productive way of writing. Alberto and Shiv got out from the darkroom at 1 am, but what happens in the dark room stays in the dark room. We submitted the judging form at 2 am (London time). Well done guys !

Saturday

Wiki day. Yutong, Alberto, Diellza, Siwat, Luis, Will, Thomas and Shiv are in RSM working hard on the wiki. Josh has probably FOMO and is calling either Diellza, Yutong ir Alberto every 5 min. We got out from the library at 4 am.

Sunday

Another intense wiki day. Also, more plate reader experiments ! We are characterising the MelA construct with more biological replicates.

Monday

Tuesday

Everybody is working very hard to finalize data and writing/polishing texts. Also everything is being uploaded on the wiki. All night in the library with everyone and then the final year students.

Wednesday

Still in the library on the wiki. The final year members of the team go to graduation completely knackered. Today is the wiki freeze.

Wednesday

We are off to Boston for the Giant Jamboree. We arrive around 6pm at the hotel and go to a local pub for dinner while watching Red Sox.

Thursday

The weather in Boston is amazing, but we decide to spend the entire day in the hotel working on the presentation.

Friday

Tomorrow is the presentation day. Will, Lidia and Siwat are practicing very hard for the last minute.

Saturday

Today is the presentation day, we are the last to present. Our presentation room is packed full of people, not only all the seats are taken, but the floor was filled too. We hope that all of you love our presentation and find our project cool.

Sunday