Team:INSA-Lyon/Biology

• WETLAB SUMMARY
• RESULTS
• NOTEBOOK
• PROTOCOLS
• 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 (Barnhart 2006). CsgA, which is the monomer of the Curli structure, can be engineered. This property constitutes the basis of our work, as we engineered the CsgA by adding one or more His-Tag motifs, famously known to be able to chelate Nickel (Hochuli 1988). 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 while chelating 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 a protocol for nickel quantification using dimethylglyoxime (DMG) that changes color from transparent to bright red in the presence of Nickel and confirmed those results using mass spectrometry.

• 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 light 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,in the WT strain, CsgA expression is controlled by the csgABC promoter but we identified, isolated and characterized a 70 base-pairs sequence that reaches higher production rates at 37°C instead of 30°C.

The principal of the construction is shown in the figure below.

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