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Team:Leicester/Project
From 2012.igem.org
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Overall project
Our project is about trying to reduce the waste going to landfill by engineering a bacteria to degrade polystyrene. Some bacteria have been found to form biofilms on polystyrene, indicating that polystyrene may be being degraded. We have several different parts to our project, including a citizen science experiment (CSE).
We intend to extract the genes involved in any pathway degrading Expanded Polystyrene (EPS), and/or develop a new pathway involving modifications to existing enzymes able to degrade aromatic and aliphatic hydrocarbons to fit polystyrene and its derivatives into the active sites. The bacteria strain that we insert these new genes into should then be able to degrade polystyrene at a higher rate than natural bacteria, and future iGEM projects at the University of Leicester could harness this bacterial strain to produce useful products.
Project Details
The Citizen Science Experiment
The CSE was used to get the public involved in trying to find bacteria that could live on, and degrade expanded polystyrene (EPS). We asked them to bury a strip of EPS in the ground and leave it for a length of time (around 2 months), to find out whether bacteria might establish colonies, thus indicating that a colony could at the very least bind to, and possibly degrade EPS. The kits were Risk Assessed, and a copy of the assessment was included in the kit, along with instructions, a strip of EPS, and a self seal bag to put the EPS into. This was all contained in a stamped, addressed envelope at a cost to the public of £2 (+50p postage if bought online via our blog to cover the cost of sending the kit to the person). We recently had our first batch of kits back, ready to analyse.
The hope is that we'll find a bacteria that has been using polystyrene as a carbon source, so we can extract the genes responsible and attach them to high expression promoters to increase the amount of protein each bacterium produces. We will also be trying mutagenesis on these bacteria to increase the rate of reaction of the enzymes involved in the pathway.
The Experiments
Plating out the returned Citizen Science Experiment kits
The protocol
The first part involves halving the expanded polystyrene (EPS) lengthways with sterilized scissors in our returned citizen science experiment (CSE) kits so that we'll still have some of each sample left should we need it. First we need to do a practice with unused EPS strips (control and test). Pour/pipette 20ml LB agar into a petri dish and allow to set. Put line down middle in permanent marker USING A RULER! Then label one side swab (for the swabbing/scraping material) and the other side strip for the EPS strip (can use other names if you prefer). Add 50 micro litres of PBS (phosphate buffered saline) to each side of the half strip of EPS and scrape with spreader, before spreading onto the left half of the agar (or side labeled as 'swab'). Try to swab any mud off the strip if possible. Then place the half strip in the centre of the other half labeled 'strip'. Leave for a week at room temperature on the lab bench and examine agar for colonies. The other half of the strip should be put back into cold storage for future use.
Day Four
(10:00 am) Started looking at the colonies and taking photos until the camera died after 11. The other 16 will have to wait until later.
(13:00 pm) Finished photographing all colony plates as the camera was charged. The next stage to make new plates which have polystyrene as the sole carbon source in an attempt to isolate the microbes from our normal agar colonies that use polystyrene, it's a way of purifying our colonies. This may take a while for the colonies to grow so our computer scientist, Emily, 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 we are 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, we 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 is the next target, and also had no effect on the polystyrene.
Day Five
(10:00 am) The polystyrene test dish is ready and has solidified so it can be plated up when we have 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, we're going to try another idea that the team had yesterday. We're going to attempt to crush the Polystyrene 'sugar' (unexpanded polystyrene beads) using either a pestle and mortar or crushing them with toughened glass beads. We'll then attempt to suspend the crushed 'sugar' in minimal medium, thus creating cloudy media, where hopefully halos around specific colonies will form when polystyrene degrading bacteria are present. Its 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 success is very slow. The pestle and mortar was producing minimal results so we 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 our polystyrene 'sugar' down, so we've 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, we've decided on 10 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 bellow to agar's surface making it easier for bacterial colonies to access the polystyrene as a carbon source.
Day Six
(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 -80oC for almost 2 hours in an attempt to freeze then smash it into a powder. It didn't work, but we have found several references that temperatures of -130-150oC 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 us 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 -180oC), 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.
Day Seven
(9:30 am) As there was no lab supervisor yesterday we are reconvening in 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 disslved polystyrene is a powder or back in beads like before.
