Team:Leicester/July2012

The group started researching methods for plating kits as well as looking for other papers that could help with the project.

(11:00 am) The group has determined the protocol after much discussion.

(14:00 pm) With the research done lab work started today. The team started to practice with unsterilised petri dishes, after getting everything needed to plate out the citizen science samples. Luria agar was collected from the store room and used to plate 5 of the citizen science experiments (CSE) in addition to a few controls using sterile polystyrene not placed in soil so to set a base line for which to compare our results with.

(10:30 am) The group continued the agar plating. As well as running a few practises to ensure our technique is correct we have the first 20 CSE kits to get through. This is the first of many big steps to take on the road to our project success.

(11:15 am) The production line of CSE plating begins.

(13:45 pm) The final lab inductions complete so now everyone in iGEM Leicester can now be in the lab. So on with the plating. 10 down 15 to go. Now our organic chemists are having a go, being guided by the lab leader.

(14:45) 17 kits down, 8 to go. The plated out kits are being left on a shelf by the bench to grow over the next few days.

(17:00) Finished plating out the last of the 25. A good days work. Now we the plates can be left to grow cultures and see what starts to grow.

(10:00 am) Started looking at the colonies and taking photos until the camera ran out of charge after 11. The other 16 will have to wait until later.

(10:30 am) Playing around with polystyrene and acetone to see what works in dissolving the polystyrene into a smaller state than EPS to put in agar as a carbon source.

Insert video here.

(13:00 pm) Finished photographing all colony plates as the camera was charged.

(13:30 pm) The next stage was to make new plates which have polystyrene as the sole carbon source in an attempt to isolate microbes that use polystyrene from among the many normal non-PS using colonies: it's a way of purifying our colonies. This may take a while for the colonies to grow so our computer scientist will run simulations of what the degradation would look like over a period of time that exceeds the running time of our project. The new plates consist of a very small amount of agar so that when this carbon source runs out they switch to the expanded polystyrene which we have melted with acetone to produce a molten polystyrene suspended in the acetone. To maintain the liquid state of the polystyrene we will use the orbital shaker provided by Heathrow scientific for which the team is very grateful. The polystyrene sludge is then placed as a layer in the glass petri dish with a thin layer of agar on and around the polystyrene to start the growth of the bacteria, but will quickly be used up on top of the polystyrene, and will support and outside colonies that spread past the edge of the polystyrene.

(14:30 pm) If this doesn't work for some reason, members of the group are testing out the dissolving power of other solvents on polystyrene. 50%, 66% and 75% Acetone (diluted with still water) had no effect on the polystyrene. Pure methanol was the next target, but this also had no effect on the polystyrene.

(10:00 am) The polystyrene test dish is ready and has solidified so it can be plated when we have grown some bacteria to see if it works as a medium.

(12:00 pm) On further analysis the original plan of a agar and layer of polystyrene covering it has proved ineffective as the polystyrene just formed a thick solid in the centre. However, the team had another idea yesterday. The idea is to attempt to crush the Polystyrene 'sugar' (unexpanded polystyrene beads) using either a pestle and mortar or toughened glass beads. Then the crushed 'sugar' can be suspended in minimal medium, creating a cloudy media, where hopefully claer "haloes" will form around specific colonies when polystyrene degrading bacteria are present. It is important to only use a low concentration of the the crushed 'sugar' as too much will mean that the halos won't form due to the slow degradation by these bacteria.

(15:00 pm) The grinding of polystyrene begins, but the process is very slow. The pestle and mortar was producing minimal results so the team switched to the grinder machine mixing our 'sugar' with glass beads to, by sheer force, crush the polystyrene down to an even finer grain.

(4:45) The grinding machine couldn't grind the polystyrene 'sugar' down, so the group has been seeing whether just adding the 'sugar' into agar may work on its own. Through experimenting with varying amounts of 'sugar' to agar (2.5% and 5% polystyrene), and varying the amount of agar used, the amount decided on is between 10 and 15 grams of agar/'sugar' mix at 5% 'sugar'. 20g of agar/'sugar' mix was just too deep as the 'sugar' just settled at the bottom or sides. At 10g agar/'sugar' mix, the 'sugar' is just below the agar's surface making it easier for bacterial colonies to access the polystyrene as a carbon source.

(10:30 am) The team that was here in the early morning had a quick briefing, and decided to get the minimal media ready. The ingredients used was exactly the same as used in the reference paper. This gave Chris some trouble trying to measure out the the salts, with some of them being only 0.001 grams per litre of water. This equates to 1 grain of salt in the container.

(12:00 pm) The agar has been autoclaved, and is now sat in the hybridizer waiting for our polystyrene to get crushed to be stirred into it. The polystyrene is proving to be very resilient and is resolving our focus to help degrade it.

(14:00 pm) The polystyrene has been frozen at -80^oC for almost 2 hours in an attempt to freeze then smash it into a powder. It didn't work, but several references were found that temperatures of -130-150^oC will then freeze it to a more brittle form that will then easily be smashed and can be used as a powder dissolved into the agar.

(16:15 pm) One of the other supervisors took pity on the team at this point so helped us out with a case of liquid nitrogen. However even flash freezing some of the polystyrene sugar in liquid nitrogen (approx -180^oC), then smashing the beads with a pestle and mortar made no change at all. Even using polystyrene dissolved in acetone then frozen will not be broken into smaller pieces, it just goes flat.

(9:30 am) As there was no lab supervisor yesterday the team returns to labs this morning to try evaporating off acetone which has actually dissolved a small amount of polystyrene. This involves using a heat block in the fume cupboard then seeing if the dissolved polystyrene is a powder or back in beads like before. Unfortunately our lab supervisor is away this morning as well so the team relocated to the computer room to do research and plan the meeting for the afternoon.

(14:00 pm) The team plus this weeks supervisor met up in one of the meeting rooms in the building. At this point all of the options were discussed and a plan was formed for the following few days.

(15:00 pm) A couple of members were taken to a microbiology lab to see if some P. aeruginosa could be acquired for use. Unfortunately the researcher using P. aeruginosa was out so the only thing that could be done was to produce some more plates for the bacteria to be placed on.

(16:30 pm) A few of the plates were finished but the team was told the researcher had come back so a few went back to his lab and he agreed to plate up the control, 5% polystyrene and 10% polystyrene plates with 5 different strains of Pseudomonas so the bacteria could start to grow and any results can be seen before plating up the CSE kits.

(10:00 am) Next the group tried out shaving EPS into acetone to see if the cloudy dissolved solution of acetone, eps and distilled water could be recreated. The solution turned cloudy but not as much as before, so it is being placed and left in the fume cupboard to see what happens.

(10:30 am) A few members of the group also tried plating out the 10g M.A. plate then sprinkling polystyrene "sugar" onto the surface rather than mixing it into the agar. This produced good results as the sugar made a layer just under the surface of the agar rather than sinking to the bottom, making it easier and more cost effective as less of the resources are needed.

Step one: measure out 0.5g of the polystyrene "sugar" on a zeroed balance using a measuring paper receptical.
Step two: make sure the agar is molten, and your petri dish is slightly warmed in the hybridiser so the small amount of agar used does not set strait away.
Step three: place the empty pre labelled petri dish onto the balance and zero. After this poor 9.50g of the heated M.A Agar into the petri dish and remove from the balance to a level table.

We noticed after the sprinkling, then agar set quickly, resulting in a thin layer of the polystyrene at the surface of the dish, which was the aim of the process.

11:00 am) Another step the group wanted to try was to gently heat the polystyrene "sugar" to see if it can be formed into more of a liquid to work with, as this could be easily spread into a thin layer.

(15:00 pm) After the group had lunch and finished up some work in the lab, we went to the Pseudomonas lab to see what had happened with the plates, and if it had grown or not. Unfortunately it had grown on the minimal media without polystyrene purely on the tiny amount of agar to make the gel set. With this new complication, the group headed back to the lab to decide on the next course of action, before stopping for the day.

(09:30 am) A slow start as the group works on lab safety and forms. These include the COSHH forms for the polystyrene sugar (that should have been done at the start but as the pentane was vented out, didn't think was needed) as well as toluene. The reason the toluene is needed is because it is an irritant, highly flammable and toxic but also dissolves polystyrene so it could be a method that works as long as the P. aeruginosa can grow on toluene. The P. aeruginosa can live on toluene as it can degrade it and use it as a carbon source before it is killed. What the team plans to do is use a minimal amount of toluene to dissolve some polystyrene and then mix the solution into the minimal media broth. The bacteria can then grow on the toluene to start then once it is quickly used up it can then work on the polystyrene as its carbon source. This method could also be used as a selection media as the toluene will kill most bacteria except for the ones that can also degrade polystyrene.

(11:00 am) While the team waited for the supervisor to be found, it was decided to go into town to look for more sponsorship. This was a fun 3 hour trip stopping in almost every shop thought of, that could have a sponsorship fund, or produced a large amount of polystyrene waste that the project is working on.

(12:00 pm) After being plagued by setbacks such as half the team turning up late, having no supervisor in the morning and severe computer and internet issues, work finally starts. The COSHH form for toluene is checked and ready, so some labwork involving both toluene and a much safer xylene can go ahead. A few more companies are phoned or emailed for sponsorship. Things are starting to pick up again.

(12:30 pm) An email has been sent to one of the staff in the genetics lab to gain access into an office for the card to book the hotel. Hopefully this can be done today and our place is ready in Amsterdam.

More research is being done on the next step of the process to isolate and remove the gene that degrades polystyrene so we can insert multiple copies into new host bacteria.

(09:30 am) A university open day, so rather than lab work in the morning the team offered to help out giving tours, had a cake sale and advertised the team and the project.

(14:30 pm) After the open day no-one could be bothered to do any work, and as it was quite late all the technicians said to leave it for the next morning.

(15:00 pm) Instead of wasting the rest of the day, the group doing tours decided to carry on filming for the rockethub video and produced quite a few out-takes as a thank you for people who sponsor the team.

(09:30 am) A university open day, so rather than lab work in the morning the team offered to help out giving tours, had a cake sale and advertised the team and the project.

(15:00 pm) Like yesterday it was too late in the day to start working so the team finished videoing for the rockethub so it can be stitched together and edited, then posted on the rockethub page.

(12:00 pm) The lab technician plated up some E. coli for the team to use. This is for one of the controls to see if the nutrient broth and polystyrene, or dissolved in toluene works.

(10:30 am) The team starts by creating the control plates with E. coli. The 3 plates created are: plain mineral media, and two of the minimal media with 5% polystyrene sprinkled on top. The E. coli is being plated on the plain media as well as one of the polystyrene sprinkled plates.

(11:20 am) With the controls plated up and placed in the warm room, testing on toluene can start to see if it creates a viable media when dissolved polystyrene is mixed with the media.

(12:00 pm) After a huge amount of walking and safety introductions the team could finally start working with toluene under the careful eye of a supervisor.

(12:30 pm) The toluene did not work. It dissolved the polystyrene perfectly, however why trying to mix it into the liquid media it precipitates out into a frog spawn like mixture.

(14:00 pm) Back to the drawing board then for the team, as new ideas need to be made to produce a viable media to grow and select for polystyrene degrading bacteria. One possible solution is to have a liquid media with nutrients in, but this still has to be tested.

(10:30 am) As a group we decided to start an hour later today as there wasn't much lab work to be done. To start off, the team had to re-suspend the liquid media as some of the salts had crystallized out of suspension. Once this was done by a combination of vigorous shaking and microwaving, the team could start making 7ml solutions in 50ml corning tubes. This allows the tubes to be spun in the orbital shaker at about 250rpm so the polystyrene doesn't settle at the bottom of the tube but is mixed throughout. The corning tubes were labelled with each strain of P. aeruginosa that are plated up as well as a control for both plain nutrient broth as well as nutrient broth/polystyrene mix where there is 5% of polystyrene in each tube.

(12:30 pm) With all the tubes filled with the correct media they were taken over to the MSB where they could have the bacteria put into the suspension and left to start growing. With this done the work for today is done.