Team:Groningen/Project

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Heavy metal scavengers with a vertical gas drive

Introduction:

Human health and the environment are endangered by heavy metal pollution in water and sediment. To improve purification strategies a metal selective microbacterial cleaning system was designed. The system comprises uptake, sequestering and metal sensitive buoyancy. All subsystems are interchangeable, which makes it suitable for almost any metal cleaning assay. For this project the modular system was focused on arsenic accumulation, using Escherichia coli as a chassis organism. Arsenite and arsenate are imported by GlpF, a aquaglycerol porin from E. coli. Intracellular As(III) and As(V) are sequestered by fMT or ArsR. These proteins were used as the accumulation modules. Since E. coli does not have a buoyancy system, the polycistronic gas vesicle protein gene cluster from Bacillus megaterium, GVP, was used. The arsenic promoter from E. coli, pArsR, is regulated by the negative transcriptional regulator ArsR. GVP, under regulation of pArsR, was used as the metal sensitive buoyancy module.

Results:

All modules were cloned according to the BioBrickTM Standard Assembly 10. The synthetic gene GlpF , was successfully cloned into a synthetic operon, with fMT. The GVP cluster, with a ten times repeat sequence, was successfully cloned downstream of the pArsR promoter. These two subsystems were transformed in E. coli to complete the system. The system and its subparts were tested using several assays. Accumulation was tested by an uptake assay, however, since no reproducible results were obtained, the functionality of the accumulation module could not be determined from these data. Arsenic uptake was examined using a metal sensitivity assay. The E. coli strain overexpressing GlpF showed a decreased final cell density upon induction with As(III), suggesting functional expression of the transporter. The metal sensitive promoter pArsR was tested using a fluorescence assay. This showed a 2.3 fold increased activity upon induction with 100 ┬ÁM NaAsO2. Buoyancy was tested by a sedimentation assay. Enhanced buoyancy was shown for the buoyancy module and the complete system, though no difference of the buoyancy phenotype could be observed upon addition of the accumulation module. Cells cultivated in aerobic conditions showed improved buoyancy compared to cells cultivated in semi-aerobic conditions. Expression of gas vescicles was shown by electron microscopy. An interactive computer model was made for the whole system, with which the modules were further characterized. With the model, import rates of As(III) at different initial extracellular arsenic concentrations could be determined. Also the influence of different parameters on the accumulation factor, the ratio between bound and unbound arsenic, was calculated. The model also allowed qualitative determination of the regulation of pArsR by various expression levels of ArsR. Furthermore, the volume fraction gas vesicles in the cells needed for buoyancy, for several sizes of the gas vesicles, was computed.

Conclusion:

The metal selective microbacterial cleaning system for arsenic was shown to be buoyant and the buoyancy module and uptake module were shown to work individually. For a better determination of the system an accumulation assay need to be redone. It was shown here that the system has potential as a cleaning system for arsenic. As mentioned earlier this modular system can also be implemented in cleaning of other substances. Literature research showed possible modules for copper, zinc, mercury and even gold. So not only cleaning water and sludge but also mining rare metals could be functionalized using this system.