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

Making Polystyrene selection media

Polystyrene selection media (or polystyrene minimal media) is made first by making up a minimal broth using the recipe from (Atiq et al.,2010) by combining [insert recipe here]. Melt 2% agarose to the broth to solidify it after cooling using a microwave, and heat untill the agarose is completely melted/dissolved. Allow media to cool by keeping it at 60 degrees C untill needed. To make a selection plate, use 95% media and 5% polystyrene 'sugar' (polstyrene beads before they've been expanded in the manufacturing process). We suggest 10 grams as a total mass of minimal media and solution. You should first pour out the media, and before solidying, sprinkle the polystyrene on top, to prevent the 'sugar' from sinking to the bottom thus allowing bacteria colonies to access the polystyrene as a carbon source. The agarose is the only other carbon source in the plate (except obviously the plate itself), so any colonies that grow should be utilising polystyrene as a carbon source, or at least have evolved pathways to deal with the toxic effects that a long chain hydrocarbon could cause bacteria (such as interferring with the cell membrane, due to the hydrophobic nature of parts of both)

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 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.

After letting the organisms grow for a few weeks, select some vigourously growing colonies and spread them out onto a polystyrene minimal media plate divided into 6 sections, where each selected colony is allocated its own segment. Leave at room temperature or at 37 degrees C, checking each day. The bacteria that grow are likely to only grow very slowly, so it could be a month or more until you see a visible colony, due to the low polystyrene degrading rates of bacterial enzymes. If a colony grows, extract some, add the sample to luria broth, and grow overnight on a shaker at room temperature or 37 degrees C. Boilate the colony by boiling the bacteria in distilled water for 10 minutes, before centrifuging at 13,000 RPM for 5 minutes to remove cell debris- the supernatent should contain DNA, which you can then add to a PCR mix and amplify overnight

The next step

Results