Difference between revisions of "Part:BBa K2123203"

Latest revision as of 08:08, 28 October 2016

Mer Operon Device

Overview

This composite part was developed to turn available all mer operon device, related to Hg metabolism.

Structure and mechanism:

This part is constituted by MerA, MerB, MerR, MerP and MerT.

MerA codifies the enzyme mercuric ion reductase, which catalyzes the reduction of mercuric ion Hg(II) to its volatile and non toxic form, Hg0. The active site of this enzyme has four cysteine residues that engage in mercury binding. The C-Terminal site is responsible for mercury catching from solution and delivering it to the core. This gene is the heart of mercury resistance in bacteria and its cytosolic protein works alongside a NADPH molecule, which offers the reductive power to reduce Hg(II) to Hg0.

MerB wides the range of mercuric forms that bacteria can be resistant to. MerB is translated into organomercurial lyase, an enzyme able to break the bonds between Hg and an organic radical, releasing Hg(II) to be reduced by MerA. This step is essential to fully sanate mercury contamination, since organomercury can be bound to living tissues and be biomagnified through food chain till it reaches humans.

MerR is the regulatory gene with its own promoter. MerR codifies the regulatory protein that binds the promoter region of the operon, in such a way that the recognition site of the promoter remains inaccessible to RNA polymerase, thus the operon's genes aren't translated. When mercury enters the cell, it binds to the C-terminal domain, causing an allosteric change on the protein's structure that propagates to N-terminal site, which is covering the promoter. This change causes the regulator to unwind DNA strand, allowing RNA polymerase to recognize the promoter and start the operon's functioning.

Mer P is a periplasmatic transporter protein, able to capture mercury organic or inorganic forms in periplasmatic space and deliver it to the membrane integrate protein, encoded by MerT gene. A pair of cysteine residues take mercury and its sulfer atoms ruptures the bonds between mercury and other binders, whose charge may be repeled by bacteria.

MerT protein, then receives mercury from MerP, at its first transmembrane helix. A pair of cysteine residues forms a complex with one cysteine from MerP, constituting mercury's deliver system. Then, another pair of cysteine, lying at cytoplasmatic face of MerT, receives Hg from the first helix and gives it directly to MerA protein, coupling mercury transport to the reduction mechanism.

Usage, Methodology and Experiments

The first step to characterize this part was testing its Hg resistance and bioremediation with and without MerB gene, as represented below, through an inhibition zone.

It has been use a 10 times concentration variation (20mg/mL, 200µg/mL and 20µg/mL) of HgCl2 in LM (Luria-Bertani variation with half salt) solid media, adding 10µL of mercury chloride solution on its paper disks. The samples were inoculated in triplicate and incubate in BOD at 37°C for 2 days. The results are shown below.

As we can analyze in the figure above, our construction with MerB gene, increasing mer operon spectrum, had a smaller inhibition zone (nearest to the disk), growing better in Hg conditions, with clear difference from other samples (control and mer operon without MerB). As we can see in the graph, measuring inhibition zone length, our construction with MerB had 30% reduced it!

On the next mercury chloride concentration, as shown on the figure below, our construction with MerB gene continued with a smaller inhibition zone, growing even more nearest to the disk!

In 200µg/mL of HgCl2, our construction with MerB gene reached approximately 60% of inhibition zone reduction, one more time enhanced in contrast to genetic circuits only with MerA. Now… the “Grand Finale” experiment in 20µg/mL, presented below!

In 20ppm of HgCl2, our construction with MerB was totally resistant and don’t had any inhibition zone, showing its potential in bioremediation process, metabolizing all the available mercury! Sequence and Features