Our project was born out of the ambition of having a positive impact in society. We conducted extensive research on different types of waste and their geographical, societal and environmental impact and found that, for several types of waste, solutions within synthetic biology were already being developed. Further research pointed towards the source of a huge amount of waste that typically accumulates in landfills: the textile industry (read more in the Description section). Figure 1 illustrates the Design-Build-Learn cycle, which we have implemented when developing our project.
Figure 1. Graphic summary of our Design-Build-Learn system. We started by
designing our constructs and
transform them in E. coli. We built our strain and planned our implementation. However, when testing
this in silico and receiving feedback from our stakeholders, we realized our approach was not optimal.
Therefore, we re-designed our project to overcome the problems that we were presented with.
Research and imagine
We started gathering information from companies, municipalities and fashion actors to comprehend the full picture and figure out why textiles are not recycled to a greater extent (read more in the Integration section). We found that the main problem is the blending of different materials. For example, in jeans, cotton cannot be recycled due to the presence of the synthetic fibre Elastane. Digging deeper into this issue, an idea started to form. We imagined a biological degradation of the Elastane fibre, by engineering a strain that would consume the elastane fibre out of the textile.
Design
To realize our idea, we designed two gene blocks containing four and five genes respectively. This design was based on being able to make three bacterial strains; one that degrades the soft segment of the fibre, one that degrades the hard segment and finally one strain that could be transformed with both gene blocks. The reasoning behind these two gene blocks was that it might be of industrial interest to degrade the soft and hard segments separately, as the final products are different and might be usable for other purposes. Our original implementation design was to grow the bacteria in a bioreactor using the plastic fibres as a carbon source. One of the reasons behind this was also our ambition to add value to the recycling process: theoretically it should be possible to engineer our strain further to produce a chemical, such as succinate, from the plastic degradation.
Build, test and learn
We built our constructs and transformed them into E. coli, but no transformants survived in the selective media. This was likely due to some assembly problem, maybe the plasmids turned out to be too large or our Gibson mix simply could not assemble that many fragments at once. A discussion on how to move forward after the transformation also emerged. When planning activity measurements for each individual enzyme, it came to our attention that there was no easy way to purify them.
While we were conducting the first experiments, the modelling team started simulating the growth of the recombinant bacteria in different carbon sources, to test whether they should be able to grow on the plastic substrates and potentially produce useful chemicals (read more in the Dry Lab section). From the genome scale modelling (GEM), we found out that the yield for E.coli growing on polyurethane, PEG, and PET, which are the plastic fibres that conform elastane, was quite low. Furthermore, according to our simulations it would also be hard to engineer the strain to produce a chemical.
Simultaneously, as we progressed with Human Practices and gathered more information from stakeholders, we learned that having a bacterial strain that degrades the Elastane fibres might not be the best option. First, such an implementation would be hard to fit in the current recycling infrastructure that many companies already have. Second, cultivating bacteria on a large scale outside of laboratory environment comes with several issues that might render the process ineffective. We also considered ethical aspects of our project in collaboration with the iGEM team Lund (the SynthEthics initiative) and found that using the bacteria immediately in the textile brings ethical concerns regarding the spread of GMO.
Improve
Based on both the results of our simulations, the ethical concerns and the advice of stakeholders, we improved our design by building his-tagged block segment plasmids instead. The new design meant having each gene in its own plasmid, and each plasmid being transformed into its own strain. Furthermore, six histidine residues were added downstream of the gene, before the terminator sequence so that the enzymes could be purified. The advantages proved to be numerous; we could now work with the enzymes at different stages in the laboratory process (as soon as one transformation was successful we could proceed with screening and protein extraction for that enzyme alone), they could be purified and measured individually (read more in the Measurement section) and the assembly included fewer fragments, which greatly facilitated the assembly.
The Proposed Implementation was also edited according to our findings during this second round of research. We went from aiming to produce Elastane-eating bacteria to producing an enzymatic cocktail, which could be produced entirely within laboratory environment. Only the GM enzymes will be used in recycling facilities, an option that was preferred in both ethical discussions and by industrial actors. Furthermore, it will be easier to fine-tune the enzyme activity to achieve an efficient degradation. The integration with the existing recycling network is also favoured, since it is easier to sell an enzymatic cocktail than a bioreactor. The design is also more convenient for future projects, since changing the composition of the enzymatic cocktail by adding enzymes that act on other plastic fibres would expand the range of plastics that could be degraded. We believe that our methodology: using in silico simulations to test the efficiency of the process, researching the available recycling infrastructure and asking the different industrial actors for feedback, has been an efficient tool to comply with the Design-Build-Test-Learn principle and has helped us to re-imagine our project in order to be more realistic in the lab and to fit better into society’s needs.
