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Team:Leicester/Project
From 2013.igem.org
Go Leic-styRRRene!
Polystyrene is a multifaceted material - light, largely chemically and biologically inert, and easily shaped, but also a highly visible pollutant that locks up valuable hydrocarbons. In 2013 the Leicester iGEM team are retaining their focus on polystyrene but will diversify our approach. We have three streams involving recycling, re-using and re-purposing polystyrene.
Recycling – This stream involves building on the work of the 2012 team in environmental prospecting for genes involved in polystyrene degradation. Currently we have isolated organisms from environmentally exposed polystyrene, and are progressing in our aim of adapting the toluene degradation pathway of Pseudomonas species.
Re-using - Consumer 3D printers are now a reality and use a variety of thermoplastics (PS and ABS, for example). While the technology is constantly finding new uses, most involve using virgin plastics, which is not good for the environment. Recycled polystyrene can be used for 3D printing, but has an additional advantage as it is soluble in limonene (an environmentally friendly solvent), but ABS is not. Complex 3D printed shapes require removable support structures - one solution is to print the supports using PS and the object in ABS, with the PS being removed by limonene dissolution. We are adapting limonene biosynthesis biobricks developed in previous iGEM competitions to enable genetically engineered machine biological "finishing" of 3D printed objects.
Re-purposing (DNA!) - Expanded polystyrene (EPS) is a great insulator, used by the construction industry to make our homes warmer, using less energy. EPS insulation is required to be flame retardant, which is currently achieved by incorporating environmentally polluting halogenated hydrocarbons. Recently DNA has been shown to be an effective flame retardant, but is expensive to produce. We are building a genetically engineered machine with inducible endoreplication (over-replication of DNA). This should yield DNA cheap enough to burn and when added to EPS make it flame retardant and environmentally friendly.
Recycling
The first of the three streams was focused on amplifying and modifying the genes of the tod operon found in the Pseudomonas putida F1 strain. The dioxygenase enzyme encoded by the tod operon degrades toluene which has a similar structure to the styrene monomer of polystyrene. Hence the main objective was to modify the dioxygenase genes responsible for toluene degradation so that polystyrene can fit in the active site of the enzyme.
The todC1C2BA genes encode the toluene dioxygenase (TDO) enzyme which is capable of oxidizing more than 100 substrates albeit the catalytic oxygenase is encoded only by todC1C2. Modelling carried out by the Leicester 2012 iGEM team using the programme Pymol, suggested that the gap in the active site of the TDO enzyme could be widen to fit polystyrene. They modified amino acids present in the gap by reducing their side chains in order to widen the gap that could allow polystyrene into the active site. The residues that they modified were Met220 -> Ala220, Val421 -> Ala421, Tyr422 -> Leu422 and Tyr266 -> Val266.
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Modified Toluene 2,3-dioxygenase enzyme with a similar view to the image above. Again, the pink residues are the active site residues. The white residues are those that have been modified. (left) The polystyrene substrate has been omitted for clarity, but note the gap is much bigger now (modified from 3EN1). (right) The polystyrene is present here: note the very tight fit into the gap
A side view of the modified enzyme. Note that the terminal phenyl group is in the active site
This view is just of the toluene 2,3-dioxygenase active site (blue), overlapped with the modified version (pink). Notice how similar the active site shape is, which means that potentially, the active site could still catalyse the dioxygenase reaction.
The Pseudomonas Putida strain F1 was grown in overnight broth. Miniprep procedures and gel purification were used to isolate and purify bacterial DNA. Polymerase chain reaction (PCR) was then used to amplify the tod C, X, F, G and B genes as well as the 16s rRNA. Thus we were able to confirm that we had the correct Pseudomonas strain using 16S rRNA gene sequencing. Therefore we proceeded to carry out a fusion PCR on the tod F, X and B genes.
These 3 tod genes were made into biobricks which were ligated to the iGEM supplied backbone (pSB1C3). A restriction digest was performed on the fusion PCR products (biobricks) and pSB1C3. Each of the biobricks were subsequently ligated to pSB1C3 and transformed into highly competent cells (DH5α). Some of the resulting colonies were then incubated in overnight broth. The isolation and sequencing of their plasmids confirmed that the genes were successfully cloned into the backbone containing the restrictions sites as stipulated by iGEM. In addition, double digestions were carried out to confirm the absence of mutations.
The gel picture shows the result of the double digestions. Well 2 is the digestion with EcoRI and PstI, well 3 is the double digestion with XbaI and SpeI. This shows that the restrictions sites are present in our biobricks.
Re-using
The second stream will be re-using polystyrene for use in consumer 3D printers. Most 3D printers need to print a support structure before printing the job in hand. The support material is then dissolved and disposed. We purposed that recycled polystyrene could be dissolved using the solvent limonene. The iGEM registry already has a limonene biobrick. The team worked with this biobrick to try to produce limonene, using bacteria to synthesise it more cheaply.
Limonene is a colourless hydrocarbon and it is present in most citrus fruits. The natural sources (lemons, oranges, etc.) produce the D-limonene, (+)-limonene, which gives citrus fruits their unique smell. It is widely used in food manufacturing as flavouring, it is also common in products such as tooth paste and its is increasingly being used as a solvent.
The 2013 iGEM Leicester team carried out experiments to show that limonene it is an effective polystyrene solvent. We were able to estimate that a 50% solution of limonene dissolves 8cm x 5cm x 2cm block of polystyrene in 9 minutes.
Our idea was to characterise the limonene biobrick (BBa_K118025) created by the Edinburgh 2008 iGEM team. Firstly we shuffled the biobrick into the pSB1C3 backbone. The reason to introduce the pSB1C3 backbone, is to separate the colonies that have incorporated the limonene biobrick, because those would be the ones present on a chloramphenicol plate.
Firstly we digested the backbone and the biobrick with EcoRI and PstI. Digestions were ran on a gel to confirm expected fragment sizes. Quick stick ligase was then used to ligate both genes with corresponding sites. Ligated DNA was transformed into E. coli and cells were grown overnight. Colonies that were present on the chloramphenicol plates, had ligated plasmid incorporated. To confirm that, overnight broths from those colonies were made and the mini prep was done. The isolated DNA was then cut with EcoRI and PstI again and fragment sizes representative of the backbone and biobrick were visible by gel electrophoresis.
Limonene biobrick supplied had a Lac operon preceding limonene synthase genes. We tried inducing the production of limonene, by growing bacteria with the incorporated ligated DNA on chloramphenicol and IPTG plates.
To detect the presence on limonene on the plates a volatile organic compound (VOC) was performed in the Chemistry department. The equipment used for this assay is a Proton Transfer Reaction- Time of Flight Mass Spectrometer. This instrument detects protonated compounds, for example limonene has a molecular mass of 136 therefore we would like to see its protonated mas channel at 137 and a fragment at 81.
The spectrometer graphs below, shows that there is no protonated mas channels at 137 and a fragment at 81, which suggested that no limonene was synthesised. Further estastical tests were carried out using R programme to determine if limonene was detected. Fistly, we had to stablish if the data had a normal or non-normal frequency distribution. Exploratory data analysis (such as histograms) showed that the data obtained from all 9 samples were positively skewed. This normal distribuition was confirmed by Shapiro-Wilk normality test.
The non-parametric test used included; Spearman correlation and Kendall tau tests.
Samples 1,2 & 3 = RFP plates
Samples 4,5 & 6 = 10.2 plates
Samples 7,8 & 9 = 5.1 plates
Correlation was made between each of the RFP plates and the limonene biobrick/pBS1C3 plates in the following order;
a) Samples 1, 4 and 7
b) Samples 2, 5 and 8
c) Samples 3, 6 and 9
After carrying out both tests (Spearman and Kendall) on groups a,b and c, p-values were < 0.05. As shown below (Spearman and Kendall respectively);
a) Between 1& 4 - p-value= 6.412e-10 and 2.53e-07
Between 1 & 7- p-value = 9.82e-13 and 4.558e-08
b) Between 2 & 5- p-value = 1.534e-09 and 3.1e-07
Between 2 & 8- p-value = 3.579e-12 and 5.655e-08
c) Between 3 & 6- p-value=8.359e-13 and 3.81e-08
Between 3 & 9 - p-value=3.566e-12 and 7.004e-08
Thus the null hypothesis (There is no correlation between the RFP plates and limonene biobrick/shipping vector) was rejected.
In conclusion, it seems that the stats tests cannot detect limonene expression too! Hence the correlation between the RFP (control plates) and the limonene biobrick plates. This suggests either a fault in the limonene biobrick or a disfunctional promoter. Unfortunately due to time constraint we were unable to run further testing to determinate the cause of the lack of limonene synthesis.
Re-purposing
Finally, expanded polystyrene is widely used as insulation in the construction industry. In some applications the insulation is required to be flame retardant, which is achieved by incorporating halogenated hydrocarbons – another environmental pollutant. DNA has been shown to be flame-retardant (link the paper and the video); however it is very expansive to produce. We proposed to build a genetically engineered machine to cheaply generate DNA for incorporation into flame retardant polystyrene. Firstly it was determined that polystyrene is indeed highly flammable (add video). Secondly we wanted to determine if coating polystyrene with DNA would change its fire retardancy in comparison to a non-coated polystyrene block.
We used Herring sperm DNA at a concentration of 13.4 mg/ml. The DNA was mechanically worked with to make it more spreadable, first by sonication, which did not seem to change viscosity of it and then by shearing through a needle, which made the DNA significantly more spreadable.
Our first combustion tests were carried out using paper-thin strips of polystyrene coated with DNA, comparing with polystyrene strips that were only coated with water (add video). As it can be seen I the video it seems that the addition of DNA aided the flammability of the polystyrene.
A second test was carried out using blocks of polystyrene made with DNA incorporated into it. Two blocks were made one with DNA and one without. As it can be seem in the videos (add them) comparing to regular polystyrene, we were able to determine that the polystyrene sugar supplied to us had HBCD incorporated to it.
Unfortunately due to time constraint were unable to aquire polystyrene sugar without HBCD to carry out further tests. For future reseach, more tests have to be carried out with polystyrene with no HBCD and also the cotton experiment should be replicated, to determine…
