PETALUTION’s design has been driven by meeting two major design criteria:
In the sections below we discuss how we designed our heavy bioremediation device, heavy metal biosensor and PET biodegradation device. We also discuss how they meet the design criteria for PETALUTION and how they work.
PETALUTION being cell-free is fundamental to our project, as the current restrictions on live GMOs prevent cell-based technologies from being used in real-world applications. Therefore, our biosensor design is cell-free, however, cell-free biosensors also have several advantages over cell-based biosensors (Table 1).
|Cell Free Biosensors||Cell Based Biosensors|
|Can use simple linear DNA and PCR products or gene could be cloned separately||Requires time-intensive cloning and transformation protocols.|
|No growth-dependant variability due to no living cells||Can have variability due to growth-dependence|
|Resistant to toxicity||Susceptible to toxicity (Including heavy metals)|
|No cell wall/membrane present so no permeability limitations||The cell wall/membrane can cause permeability limitations on the response time of the biosensor.|
Table 1. Shows the differences between cell-based and cell-free biosensors
We were inspired by Edinburgh’s 2020 iGEM team (Finding NEMO) design for their cell-free transcription-only biosensor. Their design only required the transcription of fluorescent RNA aptamers which then bind to a fluorophore to produce a readable output. The biosensor only requires T7 RNA polymerase, chemical energy from adenosine trisphosphate (ATP) and NTPs to produce this readable output. From this design they were able to produce an arsenic biosensor which could detect arsenic at levels as little as 7µM and could produce a fluorescent output within 15 minutes. This short response time is caused by the design not needing ribosomal translation which is often the rate-limiting factor in biosensor design . These promising results allowed us to expand upon their design so that we could detect more metals like mercury, lead and cadmium and test a variety of different fluorescent RNA aptamers.
The biosensor itself is a linear piece of double-strand DNA made up of five distinct parts (Figure 1):
The transcription of the fluorescent RNA aptamer is controlled by a transcriptional repression mechanism. This is where a protein that binds to a promoter will block subsequent binding of an RNA polymerase. In our design this is induced by heavy metal transcription factor which can bind to the heavy metal transcription factor binding site downstream of the T7 promoter; this will impede the function of the T7 polymerase. However, if there is a metal ion present in the reaction then the heavy metal transcription factor will then leave the linear biosensor and bind to the metal ion, this allows the RNA aptamer to be transcribed. Once this RNA aptamer is bound to a fluorophore it will produce fluorescence, this can be used as the readable output (Figure 1).
For transcription factor design we found well-characterised metal sequestering operons: MerR, PbrR and ArsR [2-4]. These operons all bind our metals interest: mercury, cadmium, lead and arsenic as these are the most common pollutants in Ghana’s waste . We used the sequences of these operons to find the promoter’s which were then used as the transcription factor binding sites for the linear biosensor. We then took the transcription factors genes from these operons and were assembled into an expression cassette. These expression cassettes were then transformed into competent cells. These cells could then be lysed to obtain a cell lysate containing the heavy metal transcription factor which was used for the biosensor reaction.
We decided to use the MerR transcription factor for mercury (BBa_K4390004), a mutated MerR for cadmium (BBa_K4390003) as it has shown better binding to cadmium than wild-type MerR , PbrR for lead (BBa_K4390005) and ArsR for arsenic (BBa_K4390002). All of the expression cassettes were composed of the constitutive promoter J23100 (BBa_J23100) and a ribosome binding site (BBa_B0034) upstream of the transcription factor and the weak synthetic L2U2H09 Terminator (BBa_K4390001) downstream of the promoter. The only parts we changed were the transcription factor genes depending on what we wanted to express. All the constructs were assembled using joint universal modular plasmids (JUMP)  and were assembled into a JUMP Level 1 vector plasmid for expression in E. coli TOP10.
Our constructs contain a double-strand DNA encoding the upstream promoter which controls the transcription of the downstream signalling component, when this component is transcribed, it will activate the fluorescence. The design is driven by a T7 RNA polymerase which uses the strong T7 promoter which can then transcribe the RNA aptamer surrounded by the F30 scaffolds.
We designed a variety of different biosensors depending on which metal we wanted to detect, which only required changing the transcription factor binding site (See Figure 2). We also decided to use a variety of different RNA aptamers to see if there were differences in the fluorescent output when the different RNA aptamers were bound to the DFHBI fluorophore to see which produced the strongest output. We decided on using Squash, Broccoli, iSpianch and Spinach 2 as these have all been shown to bind to DFHBI and produce fluorescence [2,8].
For our bioremediation device, we needed to make it cell-free and biosafe as well as sustainable; to match our vision for PETALUTION. Designing our device to be cell-free would mean we avoid the release of live GMOs into the wild and are not prevented by the restrictions on GMOs that prevent current cell-based technologies from being used in real-world applications. By also using renewable, biodegradable and non-toxic materials like hydrogels for our design we ensured our device is sustainable and poses no harm to surrounding ecosystems.
Hydrogels are rapidly gaining recognition as a more suitable platform for protein immobilisation than more traditional methods like silica bead immobilisation. This is due to their biocompatibility, robustness and, depending on their composition, their ability to host a wide variety of covalent (e.g. click chemistry, SpyTag-SpyCatcher) and non-covalent (e.g. epitopes and other ligands) molecule-capturing methods . When compared to silica beads, which few peptide tags or compounds compatible with protein modification confer strong affinities for, suggested that hydrogels were the better option for our design. Biopolymer-based hydrogels are also biodegradable and renewable whereas silica beads are a non-renewable material. This is why we selected biopolymer-based hydrogels as the matrix to construct our heavy metal bioremediation device. On their own, hydrogels are capable of absorbing metal ions , so to enhance their metal-capturing capacity we embedded a heavy metal-chelating protein into the hydrogels.
Metallothioneins (MTs) are a group of small (6-8 kDa) promiscuous metal-chelating proteins that have an affinity toward a wide range of divalent cations including Zn(II), Ni(II), Pb(II), Hg(II) and Cd(II) and trivalent cations like As(III) [11,12]. Furthermore, one MT molecule can bind 6-9 metal ions . This is because they are rich in thiol groups (high cysteine content) that form complexes with metal ions. MTs are dominated by Cys-Cys, Cys-X-Cys and Cys-X-X-Cys metal-binding motifs . Their high metal-binding capacity and affinity for heavy metal ions when compared to other proteins like phytochelatins which have a very narrow binding specificity and are only able to bind 1 to 2 metal ions  are why we chose MTs as our candidate protein for our cell-free heavy metal bioremediation system.
The MT sequence we used comes from the blue mussel species, Mytilus edulis, due to the notoriety of mussels for their heavy metal bioaccumulation and therefore, use as natural heavy metal pollution indicators in marine settings  which is the setting we plan on using our bioremediation device.
For our bioremediation device, we created ‘3C hydrogels’ (See Figure 3). They are named ‘3C hydrogels’ as they are made of three components:
CMC was used as it is a cellulose-based compound which is earth-abundant, inexpensive and sustainable, it therefore meets all the design criteria for PETALUTION. However, CMC polymers alone are soluble in water due to their hydrophilic carboxymethyl groups but mixing a solution of CMC with CA crosslinks the polymers to form an insoluble CMC-CA hydrogel matrix . The first two components are edible therefore creating no risk to wildlife and ensure the hydrogel is biodegradable.
The CBD sequence we used is of CenA, the cellulose-binding domain of the cellulase endoglucanase A from Cellulomonas fimi . We attached the CBD to the N-terminus of our MTs via a glycine-serine linker. The CBD binds to the beta-1,4 glycosidic bonds of cellulose and its derivatives like CMC , allowing attachment of the fused MT to the hydrogel matrix.
We expressed CBD-tagged M. edulis MTs in the non-pathogenic laboratory E. coli strain, BL21(DE3). Afterwards, we attached our recombinant MTs to our hydrogels by incubating the hydrogels in the lysates of the E. coli cultures expressing the CBD-tagged MTs.
Figure 3. A general schematic diagram of how our 3C Hydrogels are constructed.
The use of MTs was explored as a method to bioaccumulate toxic metals via hydrogels in the bioremediation aspect of our project. The idea of using metal-chelating proteins inspired us to also explore the potential for improving MTs binding capacity and thus their function, through Directed Evolution.
We wanted to establish a novel screening and selection system that could indicate improved MT function. We hypothesised that if we grow MT expressing bacteria in toxic metal concentrations, the bacteria will have to utilise MTs for metal chelation, critical for its survival. Therefore, bacteria that survive at an increasingly toxic heavy metal concentration, will express MTs that would be potentially interesting to characterise and study. Our experimental plan is outlined in Figure 4.
Error prone PCR can be used for generating mutant libraries by using a low-fidelity DNA polymerase (Taq M0267) which lacks 3’ to 5’ exonuclease proof-reading abilities . Increasing MgCl2 concentrations is another way to increase mutation rates by reducing DNA polymerase fidelity. To manipulate the kinds of mutations being incorporated, the concentrations of dNTPs can be varied to increase the likelihood that dNTPs present at higher concentrations are incorporated into the newly replicated strand.
We generated our MT mutant libraries using error-prone PCR under the abovementioned conditions. Based on the assumption that metal-binding capacity is a function of cysteine content, we increased the concentration of dTTPs to increase the incorporation of uracil point mutations in the transcript which would correspond to more UGU and UGC codons encoding more cysteines .
Silver is toxic to gram-negative bacteria like E. coli while also functioning as a ligand for MTs . Other MT ligands like zinc and copper have previously been used to understand MT ligand binding and binding affinity , however, we found that these metals did not exhibit usable toxicity towards E. coli strains to design a selection pressure. Silver is not a heavy metal nor is it a common water pollutant, however, it is a safer alternative to using lead, mercury, cadmium, or arsenic which are good ligands and common toxic pollutants. Thus, we decided to use silver nitrate (AgNO3), handling it carefully in light deprived conditions to ensure nothing was precipitated.
We would perform screening by plating MT expressing cells on AgNO3 containing plates of increasing concentration. We would decide the concentrations based on an experiment which checked for the Maximum Inhibitory Concentration (MIC) of untransformed BL21(DE3) cells. Colony growth was used a metric to test the validity of our proposed selection and mutant screening technique. We hypothesised that if we grow MT expressing bacteria in toxic AgNO3 concentrations, the bacteria will have to utilise MTs for metal chelation, critical for its survival.
We tagged our CBD-MT fusion proteins with an N-terminal 6xHis tag (including a TEV cleavage site) because we initially planned to purify them by Ni(II)-NTA affinity chromatography. In a more ideal research scheme, we would use purified proteins over lysates. One of our principal investigators, Dr. Nadanai, then gave us further insight on how protein purification is done and is not straightforward, including the fact that optimisations are required for purifying an engineered protein to ensure it is correctly folded and functional. Purification should also be followed by dialysis (this takes a of couple days) to remove small compounds from the eluates such as imidazole, DTT or EDTA which are strong metal chelators that may drastically affect our data if not removed from the purified extract. The entire process requires at least a month, so we were not able to carry out any protein purification due to limited time, resources and laboratory access.
Instead, we were advised to incubate our hydrogels in lysates of the BL21(DE3) cells that recombinantly expressed our CBD-tagged MTs that theoretically selectively bind to the CMC-CA hydrogel matrix. We kept the 6xHis tag on our CBD-MT fusion proteins as we had already finished our JUMP assemblies at that point and did not have adequate time do more. Even though this method is much more time-effective, it poses two major weaknesses in our hydrogel design:
Hopefully in the future, we can explore a more refined hydrogel-immobilised MT design using purification techniques.
Like every other part of PETULATION, we designed our polyethylene terephthalate (PET) biodegradation device to be cell-free and sustainable. However, we are designing a tool for the PET plastic recycling industry which is not designed to be used directly in water bodies like our biosensor and bioremediation device, but, rather by industrial plastic recycling facilities. We have therefore had to tailor our design to reflect this different use case.
Silica is a cheap and robust material that has been commonly used in the laboratory for immobilization since 2006, however, it is not a biodegradable or renewable material. Despite this, we believe that silica for this use case is better as silica has enhanced stability which increases the usability of the enzymes bound to it when compared to a biodegradable matrix like hydrogels. Silica features are therefore more suited to an industrial setting like the one we plan to use the biodegradation device in.
Through our literature search, we found two silica tags which would allow us to attach our enzymes (PETase and MHETase) to silica. The conserved E. coli L2 ribosomal protein (L2NC) bound very tightly to the silanol moiety of mesoporous silica , which provided an opportunity for the L2 fusion protein to be immobilized on the surface of unmodified silica. Our second silica tag was the Car9 protein which was originally thought to bind to carbonaceous substrates and was found to bind to silica as well . These two silica tags were also used by the Edinburgh 2021 iGEM team (Super Grinder), however, we wanted to evaluate L2NC and Car9 more systematically. To do this we attached the silica tags to the N terminus and C terminus of different PETases (Figure 6) and assessed the effect of this attachment on the solubility as well as the activity of the enzymes. For the MHETase tagging we found there was a ∼60 Å long intrinsically disordered tether structure (residues 1–25) at the N-terminus of the MHETase . Therefore, we assumed N-terminal silica tags are more suitable than C-terminus for MHETase (Figure 3).
PETase was discovered in 2016 in Ideonella sakaiensis, which can use PET as a single carbon source . The PETase hydrolyses PET polymers and produces four products terephthalic acid (TPA), mono(2-hydroxyethyl) terephthalate (MHET), and bis(2-hydroxyethyl) terephthalate (BHET), ethylene glycol (EG). PETase will mainly produce MHET from PET; the highest yield of MHET is 2.5 times more than that of TPA. TPA is much more usable than MHET as TPA can be converted into high-value products like vanillin. MHET also has an inhibitory effect on the hydrolysis of PET. However, since only a very small amount of MHET can be continued to be hydrolysed to TPA by PETase, we needed to add an MHETase to convert MHET to TPA to increase TPA yield and purity .
PETase is the only enzyme which can convert PET plastic into chemical monomers which is why it was selected for our biodegradation device. However, through machine learning techniques a more efficient PETase was discovered FAST-PETase . The FAST-PETase has five mutations PETase (S121E/D186H/R224Q/N233K/R280A) these mutations allow it to break down PET much faster than the PETase discovered in 2016. We also tested a double mutant PETase (S238F/ W159H) and a triple mutant PETase (T140D/R224Q/N233K) as we wanted to compare the efficiency of these different mutant PETases.
Increased crystallinity limits the movement/fluctuation of polymer chains and decreases the availability of polymer chains for enzymatic attack. PET molecules with low crystallinity have amorphous substrates, which are easier for the enzyme to bind to the substrate . Highly crystalline (>20%) PET molecules have crystalline substrates that are difficult for enzymes to bind to, so the yield of degrading highly crystalline PET molecules with mixed enzymes is low. Currently the industry pre-treats highly crystalline PET by first melting it at 290°C and then cooling it rapidly to reduce the crystallinity of the PET . The use of these high temperatures to degrade the PET would require lots of energy and would not match our design criteria for PETULATION being sustainable.
To combat this, we found the RolA hydrophobin protein which can be found in fungi. Hydrophobins can increase the hydrophilicity of the PET product surface, thereby increasing the affinity of the substrate for PETase and thus the release of the final product . So, we either pre-treated the PET raw materials with RolA hydrophobin or fused RolA with PETase to see if we could mitigate the effect of high crystallinity levels on PETase function and use our device at room temperature to make it a sustainable product.
After designing the silica beads decorated with PETase and MHETase enzymes we wanted to create a device with these immobilised enzymes with enzymes such that they could be used easily by a recycling facility (Figure 7). Silica beads have completed their reaction with the PET, the solution is passed through a large pore size filter membrane to separate (enzymes and reaction products) from (large impurities, undegraded PET). The initially filtered solution is then passed through a smaller pore size membrane to separate the working enzyme from the reaction substrate (TPA & EG), this step also allows the working enzyme to be recovered for reuse. The isolated TPA and EG substrates can be chemically purified or specifically taken up by engineered organisms for downstream use.