The project objective was to contribute to the development of a microbial wastewater treatment system for the detoxification of pharmaceutical waste, especially diclofenac (2-[2-(2,6-dichloroanilino)phenyl]acetic acid). Diclofenac is a non-steroidal anti-inflammatory drug which causes major environmental stress in our local area, the Baltic Sea. Additionally, the current removal efficiency of diclofenac is only 27% at our local wastewater treatment plant. Therefore, a solution is urgently needed to tackle this problem.
The plan for this was to utilize laccases, specific enzymes that are capable of catalyzing the conversion of diclofenac into less harmful derivatives. Only bacterial laccase options were considered to facilitate production of active enzymes in prokaryotic organisms. The laccases chosen for the work were CotA from B. subtilis, CueO from E. coli and Yak from Y. enterocolitica subsp. palearctica strain 7 (read more about these laccases from our Design page). These were selected as they have shown high activity in near neutral pH conditions, suitability for diclofenac detoxification, or do not yet have wet lab data from previous iGEM teams by whose projects we were inspired (Team Darmstadt 2020: https://2020.igem.org/Team:TU_Darmstadt, Team Kaiserslautern 2020: https://2020.igem.org/Team:TU_Kaiserslautern and Team Stuttgart 2020: https://2020.igem.org/Team:Stuttgart).
The laccases were to be overexpressed in the E. coli BL21(DE3) strain utilizing the pET system, which is one of the most powerful recombinant protein production systems (Mierendorf et al., 1998). The expression vector of choice was pET36b(+) as it allowed for the addition of a C-terminal His-tag to the laccases. A C-terminal His-tag was selected as it enables a simple protein purification technique while minimizing enzyme activity loss (Li et al., 2017). The produced laccases would be extracted using Ni2+ affinity chromatography after cell lysis through sonification.
Next, the enzyme efficiencies of the three heterologous laccases would be compared at conditions simulating our local wastewater treatment plant through enzymatic assays. The enzyme activities would first be determined by applying the ABTS (2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid)) assay, a spectrophotometric technique based on the oxidation of ABTS by laccase (More et al., 2011). Our plans also included measurements with LC-MS to obtain diclofenac-specific results about the produced laccases. The most suitable laccase for us would be the laccase that is found to be most active especially in regards to diclofenac in wastewater conditions.
This laccase would then be overexpressed in the model cyanobacterium Synechocystis sp. PCC 6803, which is the chassis of our proposed implementation. The expression vector for this would be the shuttle vector pDF-lac2, modified from the pDF-lac plasmid, and the expression construct transformed by natural transformation (Thiel et al., 2018). Cyanobacteria were chosen for the proposed implementation as they are photoautotrophic, thus providing a sustainable alternative for heterotrophic production platforms.
Please find more information on the entire design process and details about our chosen elements on our design page.
In the build phase of the project we assembled our expression constructs and generated our engineered E. coli BL21(DE3) strains for laccase comparison. The three laccase coding genes were ordered as synthetic DNA fragments from IDT. The fragments were designed to be flanked by the NdeI and XhoI restriction sites. They arrived in pUCIDT-Kan plasmids from which they were subcloned into the pET36b(+) expression vector received from a biochemist at our university. This resulted in three final expression constructs, pET36b-cotA, pET36b-cueO and pET36b-yak. These subcloning steps were performed in the E. coli DH5ɑ strain. Once the final expression constructs were assembled, they were transformed into the E. coli BL21(DE3) strain. The generated strains were verified through restriction digestion with NdeI and XhoI. As the ordered genes were obtained as fully verified, sequenced fragments, there was no need to confirm the final constructs by DNA sequencing, as PCR amplification was not used in expression plasmid construction.
Generation of the expression constructs for our proposed implementation chassis, Synechocystis sp. PCC 6803 was done simultaneously with the pET36b constructs. For these, another set of the three laccase genes were ordered as synthetic fragments from IDT. The difference between these and the genes that were used for the pET36b based expression constructs was only the initial restriction site, NsiI instead of NdeI, that flanked the gene. The laccase genes received in the pUCIDT-Kan plasmids were first subcloned directly downstream of the S3 RBS sequence in the pNiv plasmid (NsiI-XhoI). Another set of restriction enzymes, BspHI for pUCIDT-cotA and pUCIDT-cueO, and PvuII for pUCIDT-yak, were used to avoid digestion products similar in size. A combined fragment consisting of the RBS and laccase gene was then digested from the pNiv carrier plasmid. These were to be subcloned into the pDF-lac2 expression plasmid (SpeI-SalI) to generate the final expression constructs. Their assembly was, however, never completed as work with the generated E. coli BL21(DE3) strains for evaluation of laccase function was prioritized.
Please find more information on the build phase including gel pictures on our results page.
Once the three E. coli BL21(DE3) laccase overexpression strains were generated, and the proteins of the correct size were obtained, the aim was first to see if any enzyme activity could be observed. This was first attempted by using ABTS as substrate. However, during the initial analytical trials, we observed that the ABTS stock we had obtained was no longer useful, possibly due to oxidation and subsequent decomposition during earlier storage. To continue the analysis we decided to use an alternative substrate syringaldazine which has been previously used for laccase characterization.
The kinetic enzyme activity assays unambiguously demonstrated the laccase CotA from Bacillus subtilis was catalytically active, and able to oxidize syringaldazine. This demonstrated that one of the three enzyme targets selected for the project was successfully overexpressed in a heterologous host, purified, and confirmed functional, therefore completing one of the primary goals of the iGEM project.
The project allowed us to walk through the entire step-wise process from construct design, genetic assembly of multiple alternative expression plasmids, generation of corresponding E. coli overproduction strains, protein overexpression and purification, and finally, the characterization of the enzyme activities. As part of the process, we learned that things in science do not always work as anticipated, and that many side-tracks can be avoided by careful planning, and preparation for alternative execution plans at every stage of the project.
As an example of the practical lessons of the work, one of the three enzyme targets in this work (Yak from Yersinia enterocolitica) was not expressed in soluble form, a second enzyme (CueO from E. coli) was not catalytically active although successfully purified, while the third enzyme (CotA from Bacillus subtilis) was expressed, purified and verified to be catalytically active in vitro.
Please find more information on the learn phase on our results page.
Design in the following engineering cycle
From the beginning, the work was designed to look beyond the expected achievements in the project, and plan for the next steps. In practise, these included specific plans for:
- comprehensive kinetic characterization and optimization of the laccase reaction conditions,
- structure-based site-directed mutagenesis to improve the catalytic performance of the successful target enzyme CotA,
- completion of the corresponding cyanobacterial expression constructs and the generation of the Synechocystis sp. PCC 6803 expression strains,
- introduction of signal peptides to allow the excretion of the expressed CotA to the extracellular medium,
- further improvement of the cyanobacterial expression system by translational optimization, and
- incorporation of a molecular kill-switch that would be required for the use or large-scale confined testing of the system outside the laboratory.
- Li, L., Xie, T., Liu, Z., Feng, H., & Wang, G. (2017). Activity enhancement of CotA laccase by hydrophilic engineering, histidine tag optimization and static culture. Protein Engineering, Design and Selection, 31(1), 1–5, https://doi.org/10.1093/protein/gzx064.
- More, S. S., P S, R., K, P., M, S., Malini, S., & S M, V. (2011). Isolation, Purification, and Characterization of Fungal Laccase from Pleurotus sp. Enzyme research, 2011, 248735, https://doi.org/10.4061/2011/248735.