Our project
Hey there!
We’re really glad that you would like to learn more about our iGEM-project for 2017! But first, could I trouble you with a question?
Do you every stop to think about the choices you make in your everyday life? Today I woke up, lets confess – after several rounds of snoozing – and brushed my teeth, did a load of laundry and hurried away to the obligations of today, there among my soccer practice at six o’clock. Not once would I have stopped to consider the simple formula of cause and effect in these everyday activities of mine, if it wasn’t for being a part of this iGEM-project, because who would have thought that choices made during a completely normal day could lead to environment destruction and the suffering of animals? Not me! But the toothpaste I used, could have been a glittering one – a glitter that arises from the addition of microplastics in it, the load of laundry I did could have contained clothing in fabrics that caused release of about 700 000 microplastic particles [1] with each cycle in the washing machine and finally, maybe ran I – during my soccer practice – on a field made up of artificial grass leading to the release of about 1000 tonnes [2] of pollution particles discharged into the environment. Imagine the impact you can have on the Earths future; you are part of deciding its faith!
What we – and other people – refer to when talking about microplastics throughout this homepage, are tiny pieces of plastic – less than five millimeters long – that can be found in many health and beauty products such as hair products and toothpastes, or arise from larger plastic debris that are degraded into these tiny pieces by for instance the UV-light during a sunny day. These particles are not, to our knowledge, harmful for human health but the origin of the problem arises when we wash these off our skin or clothes. Because when microplastics find their way to the water treatment plants, the filtration systems used there are currently not capable of catching these particles, they are just too small which means that they travel on and eventually end up in our marine environment instead – the ocean, the home of about 230 000 living organisms [3].
To plankton and fishes the microplastics look like food and they are ingested by a variety of aquatic organisms, leading to their entering in the food chain. A food chain that also we humans are part of which means that microplastics in the end can end up in our own tummies. These deceitful tiny pieces of plastic can interfere with digestion and reproduction in the aquatic species and may cause physical harm, but that is not the only problem [4]. They can also leach chemicals such as plasticizers – which is a compound added to give a plastic material improved flexibility and reduce brittleness – and attract toxic chemicals such as persistent organic pollutants (POPs) – which is an organic compound that is resistant to degradation and therefore could accumulate to a potentially toxic level.
One may think that this is a recent problem but can you remember a time when your tooth paste was not glittering? According to the United Nations environment Programme, microplastics started to appear in these kinds of products about fifty years ago and just five years back this issue was still not raised and consumer awareness was low. Although in 2015, former President Obama signed the Microbead-Free Waters Act of 2015 [5] and banned plastic additives in cosmetics and personal care products. [6] Also, recent discussions regarding the realization of the UN development goal number 14 concerning the conservation and sustainable use of marine resources have shed light on the matter and during the last year several companies have taken a stand by for instance not giving out plastic bags for free in the shopping malls [7] and avoid selling products containing microplastics [8].
We, iGEM Team Lund, therefore chose, in line with the UN's new framework for sustainable development, to devote this year to the increasingly aware issue of microplastic accumulation in the ocean.
Our project focuses on designing a way to determine the presence of microplastics in water by *drum roll* implementing a genetic circuit in E. coli and utilize a logic AND-gate to create a biosensor for indirect detection of microplastics using two different molecules – the plasticizer and POPs mentioned earlier and often associated with microplastics.
So, to break it down, E. coli is the, probably, most common lab bacteria and due to that; it is also easier to work with since a lot is known regarding it. For E. coli to live its life, it has – just like all other life on earth – a genetic code, in the form of DNA, which is transcribed to RNA and then translated into proteins that are useful for the organism itself. One such protein is Green Fluorescent Protein (GFP) which, like its name reveals, fluoresces green when expressed and therefore enables an easy way to detect and measure the amount of expression of that particular protein. But for a protein to be expressed, the DNA has to contain at least 4 things; a promotor – which marks where the gene should start being transcribed, a UTR (ribosome binding site) to where the ribosome can attach and do its translation into protein, your favorite gene – encoding whatever you would like to express, and a terminator – which marks where everything should end. Also if your gene is added to the bacterium in form of a plasmid, a plasmid backbone is also needed which contains, amongst other things, antibiotic resistance that gives an advantage to the bacterium which picks up the plasmid since the cells would not be able to survive the antibiotic we will be adding unless they have this plasmid. A genetic circuit is basically inserting DNA into a cell in order for it to carry out logical functions similar to what can be done in electronic circuits – and it is commonly used within synthetic biology. In this project, the gene of interest is GFP, but it is split into three pieces and it will only glow if those pieces are connected again. The promotor that is initiating transcription of one of the GFP-parts are only activated in the presence of POP, if POP is not existing in you water sample – then the GFP-part is not obtained – but if POP does exist in you water sample, then it will form a complex with nahr and activate the transcription of one of the GFP parts. But one GFP-part is not enough to receive a glowing measureable signal. The other two GFP-parts needed is connected to the two ends of hER-alpha (which is the alpha version of the estrogen receptor) and this protein is always expressed in this biosensor, but since the two GFP-parts are on either end of it, they will only be close enough to merge if phthalates are present in your water sample. If there are no phthalates in your water sample – nothing happens and no fluorescence is obtained, but if there is phthalates in your water sample then the hER-alpha receptor will bind them and go through a conformational change which causes the two parts of GFP to be close to enough to each other to work as GFP. Well, almost – because we would of course also need the third part of GFP that was expressed if POP was present in you sample. Think of it like playing hockey – if three persons wants to play hockey (kind of like our three GFP-parts wants to glow), but they only bring the hockey stick or they only bring the puck – it will be a bit hard to play… But if they bring both the hockey stick and the puck – now it’s looking to be a great afternoon! Same applies for the GFP-parts, they will only be able to fluoresce if all three of them are connected, which they can only be if both POPs and phthalates are present in your water sample, which these molecules likely are… if your water sample contains microplastics! So now that you understand this, that all three parts must be present, then you understand how a logic AND-gate work! And most likely, you’ll also very soon be able to rock in synthetic biology!
References
- [1] https://www.theguardian.com/science/2016/sep/27/washing-clothes-releases-water-polluting-fibres-study-finds
- [2] http://www.kimointernational.org/news/microplastic-pollution-from-artificial-grass-a-field-guide/
- [3] https://www.newscientist.com/article/dn14206-how-many-species-live-in-the-sea/
- [4] http://www.pbs.org/wgbh/nova/next/earth/freshwater-microplastics/
- [5] https://www.gpo.gov/fdsys/pkg/BILLS-114hr1321enr/pdf/BILLS-114hr1321enr.pdf
- [6] https://oceanservice.noaa.gov/facts/microplastics.html
- [7] http://www.onebaghabit.se/
- [8] https://www.dagensapotek.se/artiklar/2016/12/09/apotea-stadar-bort-miljobov/