Team:UUlm/Description
Project Description Team UUlm
The inspiration for our project originated in a microbiology lecture by our PI, Prof. Dürre. We heard about tooth decay triggered by
Streptococcus mutans.
This bacterium deals with the hardly accessible apatite of the teeth by releasing acids that dissolve the substance. By forming holes and thereby increasing the surface area of the teeth,
S. mutans
can quickly destroy the chewing apparatus. As our future team leaders discussed the topic afterwards, they imagined a scenario in which a similar effect could be quite useful: plastic degradation. A great problem in that field is the high structural integrity of plastics and their small attackable surface area. If plastic eating bacteria could be equipped with the ability to excrete a solvent for the plastic, Katharina and Christoph thought, this could help them to degrade it faster. One plastic came to their minds that is especially prone to household solvents: polystyrene or PS for short. One of its solvents is acetone, which can be produced by bacteria such as
Clostridium acetobutylicum.
After a short research, they had found out where to look for PS degrading bacteria. As they searched for the topic online, they stumbled upon mealworms (the larvae of the beetle
Tenebrio molitor)
which are capable of PS degradation. Further research confirmed the thought that intestinal bacteria of the larvae were responsible for this ability. If these bacteria could be genetically modified to produce acetone, they may – just like
S. mutans
– be able to degrade their substrate better.
In 2020, the global pandemic of SARS-CoV-2 required everyone to comply with special safety precautions. One effect of these measurements was a very restricted and limited access to the laboratories at the university. Only initial pre-tests could be performed by the two students allowed in the lab. Nonetheless, important findings and experiences could already be gained. They will serve as a base for the continuation of the project in 2021. Namely one population dynamics study could be conducted on mealworms, the larvae of the beetle
Tenebrio molitor.
These arthropods possess the extraordinary natural ability of degrading PS with comparably high rates and serve as base of the project.
The population dynamics study was conducted to verify the polystyrene degrading ability of mealworms and learn about their handling. Two groups of mealworms were compared over the course of 36 days. One control group was fed with oatmeal and one test group was fed with a pure extruded polystyrene (EPS) diet. The compared parameters were the masses of five fractions: larvae, substrate (EPS or oatmeal), dead material, faeces, and pupations. The fractions were divided by sieving and manual selection with tweezers. Fig. 1. shows the masses of the separated fractions.
The total mass was measured by weighing the whole container and subtracting its empty mass. The total mass together with the substrate mass and summed up biomass (larvae, pupae, dead mass, and faeces) are depicted in Fig. 2.
From the resulting data, characteristic rates could be determined for the test and control groups. Therefore, a regression of linear area of the curves was performed to calculate the gradient. This gradient equals the respective change rates of the mass fractions. From this change rate, a relative change rate was determined by dividing the change rate through the initial mass of the used larvae, being 50 g for each group. The substrate change rate for the control group includes the change rate of the faeces, as they could not be physically divided and were measured together. Besides substrate change rates and larvae mass change rates, change rates for the total biomass were calculated by adding up the mass change rates of the larvae, the pupae and dead mass. These rates are stated in Tab. 1.
| Control group | Test group | ||
|---|---|---|---|
| Substrate change rate | [g/d] | -1.135 | -0.082 |
| Relative Substrate change rate | [1/d] | -0.022 | -0.002 |
| Larvae mass change rate | [g/d] | -2.03 | -1.321 |
| Relative Larvae mass change rate | [1/d] | -0.04 | -0.026 |
| Biomass change rate | [g/d] | -0.015 | -0.709 |
| Relative Biomass change rate | [1/d] | -0.000[3] | -0.014 |
In comparison to the relative substrate change rate of the oatmeal, the relative substrate change rate for the test group is about tenfold smaller. This small change rate indicates that even in the larvae, the degradation of PS is relatively low. Other research groups facing similar observations hypothesised that this is due to the disadvantageous unbalanced diet which only feeds the respective gut bacteria, but their energy yield is too small to provide enough calories for their
hosts.[1],[2]
This hypothesis is encouraged by the overall loss in biomass. The mentioned research groups were able to compensate the effect by adding other, more accessible nutrients to the
diet.[1],[2]
For further comparison of the data, a starvation group of the mealworms would be helpful. Also, the residual undigested PS in the faeces and biomass in general was not measured, so the actual rate of degradation is probably even lower.
Parallel to the population dynamics study, acetone synthesis plasmids based on the work of our instructor M. Sc. Teresa Schoch were constructed. The aim is to enhance the ability of the gut bacteria by genetically modifying them to produce acetone. Acetone is a solvent for PS and weakens the polymer’s intermolecular interactions. In theory, the presence of this chemical should therefore enlarge the attackable surface area for the bacteria and allow for a more efficient degradation.
The acetone synthesis plasmid carries the needed genes for the acetone production: encoding thiolase A
(thlA),the butyrate-acetoacetate CoA transferase subunits A
(ctfA) and B (ctfB), and the acetoacetate decarboxylase
(adc).
Acetone is produced from acetyl-CoA. The first step of this conversion is catalysed by thiolase A. Two molecules of acetyl-CoA are converted into one molecule of acetoacetyl-CoA. The transfer of coenzyme A (CoA) is achieved by the acetate/butyrate:acetoacetatyl-CoA transferase, which results in acetoacetate. The last step is carried out by the acetoacetate decarboxylase and is the elimination of CO2. The product of this is acetone. The pathway is shown in Fig 3.
The three genes mentioned above were obtained from two different plasmids (pJIR750_ac2t2 and pJIR750_ac3t3), which were already assembled by project instructor M. Sc. Teresa Schoch. These donor plasmids are variations of the gene constellations and gene origins on the same backbone. M. Sc. Teresa Schoch also designed the acetone synthesis plasmids for us. For those designated plasmids, a pMTL83151 shuttle vector[19] was chosen as backbone. This backbone was originally obtained from Prof. Minton, University of Nottingham. It already contains resistance genes against chloramphenicol (catP), an origin of replication (repH), genes for replication in Gram-negative bacteria (ColE1) and genes enabling conjugation (traJ). The three genes obtained from the donor plasmids were combined with a PthlA promoter and inserted into the shuttle vector. The resulting plasmids are shown in Fig. 5.
The plasmids were transformed into
E. coli
XL1 Blue MFR’. Unfortunately, sequencing the plasmids after isolating them from the transformed bacteria revealed that the targeted genes were not present. The experiment needs to be repeated to gain further information. Once the transformation succeeds, then the mutant strains can pass the acetone synthesis plasmid on to the intestinal bacteria. If they produce acetone, it can be examined if this has a positive impact on the PS degradation abilities. To learn more about the scientific work done and our findings, read
Engineering success.
To learn about our future plans and the continuation of the project as well as all other parts of the project, please watch our
Team presentation video.
As a first participating team, the iGEM competition was especially hard to join for us under the special conditions of the year 2020. We still managed to participate and gain many valuable experiences. To give something back to the iGEM community, we decided to write a
Beginner's guide
for every team that wants to join in the following years. It is a summary of all the obstacles we were facing and figuring out ourselves the hard way. Hopefully, it will provide new teams the information they need and spare them the extensive research.
As the safety precautions were announced, we decided to focus more on other important aspects of our work. The
Human practices
are such an aspect. Besides the exchange with several experts in diverse fields that encounter plastics, our special focus was on the water cycle. Under the motto “it all comes back” we investigated the close loop from drinking to waste water and were invited to the Landeswasserversorgung Langenau, Germany that provides a whole region of Southern Germany with drinking water to discuss the impact of microplastics. Many other interviews and meetings are already planned. Besides exchanging with experts, we are eager to inform a broad public audience about the topic of our research. This is important because plastics can be found in all our bodies. Besides spreading awareness and publishing what we are doing against plastic pollution, we also give tips on how everyone can reduce and avoid plastics in general. To spread this information, we maintain a series of social media channels:
As we think that science and research are key to solving all of mankind’s problems, including plastic pollution, we initiated a freely available series of fundamental life science info sheets called iGEM explained. And as children are the scientists of tomorrow, we also published a series of free online video lectures on the same topic especially conceptualised for schools to be implemented into science classes called iGEM at school.
Finally, to find out about how the plastic free future we dream about possibly looks like, read our Proposed implementation and watch our presentation video.
Team iGEM_UUlm
October 2020
References
- [1] Yang S., Brandon A. M., Flanagan J. C. A., Yang J., Ning D., Cai S., Fan H., Wang Z., Ren J., Benbow E., Ren N., Waymouth R. M., Zhou J., Criddle C. S., Wu W. (2017). Biodegradation of polystyrene wastes in yellow mealworms (larvae of Tenebrio molitor linnaeus): Factors affecting biodegradation rates and the ability of polystyrene-fed larvae to complete their life cycle. Chemosphere, 191, 979-989, DOI: 10.1016/j.chemosphere.2017.10.117
- [2] Weis M., Weis L. (2018). Tuning eines bioreaktors - Optimierung des styroporabbaus durch mehlkäferlarven. Jugend forscht