Our project is dived into the detection of cadmium waste and the remediation/recycling of cadmium ions. Therefore, the proposed implementation of the project is also split into these subtopics.
Firstly, the implementation of the chip-based detection kit for cadmium assays outside of the laboratory. Here we created a chromophoric E. coli strain based on a knockout strain of all methyl-accepting chemotaxis proteins (Tsr,Trg,Aer,Tar and Tap) originally manufactured and kindly provided by John S. Parkinson from the University of Utah (John S. (Sandy) Parkinson – School of Biological Sciences (utah.edu)). We implemented mutants of the Tsr (inactive, just there for dimerization and function of Trg) and Trg (mutant for not binding the galactose binding protein) receptors together with the ribose binding protein (RBP) that was redesigned earlier this year (Li Hengyi et al. 2022) to specifically bind cadmium ions (CdRBP1m). The additional chromoprotein Meff Blue (Liljeruhm et al. 2018) was added with the same strong constitutive promotor as the other three genes. We tested the capacity of the strain to detect cadmium residues which is more thoroughly described in the results section.
The short-term improvement to the system that we planned but didn’t achieve in time is the knockout of the native ribose binding protein as it could be a source of false-positive results due to the binding of ribose in the chemotaxis assay.
Regarding the implementation of the system a specifically made chip for the chemotaxis assay with a build in magnification system for easier evaluation and sealed storage chambers for the GMOs would be a needed addition to realize the use of these chips in the field in the confines of GMO laws. Furthermore, a shorter distance between source- and sink hole would decrease the timeframe needed to analyze the chemotaxis behavior towards the sample. This also would allow to conduct the assay in aqueous solution instead of agarose gel, similar to the original publication of the CdRBP1m protein (Li Hengyi et al. 2022).
The second part of the project revolved around the remediation strain, where we implemented hMT2 (Klaassen et al. 2009) and AtPCS1 (Zhang et al. 2019) as constitutive storage proteins for cadmium ions in the cytoplasm of BL21 E. coli cells. To import more cadmium ions from the environment we transformed the native E. coli transporter MntH with a strong constitutive promotor. Furthermore, a combination of CysP (B. subtilis) and a CysE (E. coli) mutant (Denk und Böck 1987) was implemented to aid the necessary uptake of needed sulfate and production of cysteine. Lastly to recycle the cadmium ions into solid Cd-quantum dots we added another copy of the cysteinyl tRNA synthetase (EcCARs) from E. coli under a strong constitutive promotor based on findings recent publications, that indicate the formation of reactive cysteine species, especially in the presence of the co-factor PLP (Akaike et al. 2017) (Li et al. 2019).
As a long-term improvement, the intrinsic production of the co-factor PLP (boosts EcCARs activity) in the E. coli strains is a long-term goal. There are two known pathways, which yet only achieved a slight overproduction in comparison to the normal metabolic level (He et al. 2022).
Furthermore, testing the EcCARs mutant C28A, C209A in terms of quantum dot production was initially planned. The double mutant makes the tRNA synthetase inactive regarding the connection of amino acids and their tRNAs and only leaves the active secondary active site (K73/76 and K266/269) with is responsible for the binding of the PLP cofactor and the creation of reactive cysteine species for quantum dot production (Akaike et al. 2017).
Regarding the implementation of the system, a filter system+ in the sewage system of industrial production plants with an installed UV-light fail safe would be ideal to ensure the safety of such an installation. These set-ups could be tested prior in bioreactors, regarding the effectiveness and safety of such a system in a confined environment.