Team:INSA-Lyon/Biology

• WETLAB SUMMARY
• RESULTS
• NOTEBOOK
• PROTOCOLS
• PARTS
• DATA PAGE

Our wetlab work focuses on designing a bacterial strain able to chelate as much Nickel as possible but also to adhere to a synthetic matrix for future filter design applications. To do so, we engineered a hair-shaped protein polymer located at the bacterial surface, called curli (SOURCE). CsgA, which is the monomer of the Curli structure, can be engineered by our will. This property constitutes the basis of our work because we engineered the CsgA by adding one or more His-Tag motifs, famously known to be able to chelate Nickel (SOURCE). Our project aims at modifying an Escherichia coli strain that naturally produces abundant biofilm and at the same time produces the engineered curli proteins to chelate Nickel. This way, the bacteria have the sufficient adhesion ability to stick to the filter matrix and be exposed to polluted water and chelate the environmental nickel.

Unlike most metal bioremediation projects, our solution does not rely on intracellular capture, which means we can kill the bacteria and degrade their DNA using physiochemical methods to ensure the safety of the biofilter

• We constructed and cloned a modified CsgA that has either one or two His-Tag motifs. This way, we will be able to investigate if a repeated His-Tag motif is able to chelate more Nickel or not.

• We designed and optimized two protocols for nickel chelation investigation: one based on post-centrifugation supernatent exposed to nickel and a second one based on curli purification only. Both technics use dimethylglyoxime (DMG) that changes color from transparent to bright red in the presence of Nickel.

• We explored the biofilm production of our strain and its adherence ability by using the Congo Red dye and further visualized it by Transmission Electronic Microscopy (TEM).

• We investigated the bacterial survival after increased exposure to UV lights and high temperatures. The purpose is to optimize sterilisation methods for future filter design.

• We engineered and simplified the promoter sequences responsible for CsgA production. In fact, it was originally produced using the wilt-type CsgA promoter but we identified, isolated and characterized a 70 base-pair sequence that reaches higher production rates at 37°C instead of 30°C.

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