Difference between revisions of "Part:BBa K2123202"

Latest revision as of 08:08, 28 October 2016

MerT + MerP (Hg transporters) + MerB (Organomercurial Lyase) + MerA (Mercuric Reductase) + B0015

Overview

This composite part was developed to turn available the mer operon genes, related to Hg metabolism: MerT and MerP mercury transporters, organomercurial lyase (MerB) and mercury reductase (MerA), without the MerR regulator, as you can see below.

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With this part, you can switch the mer operon promoter sequences, regulating (or not) by what you want. MerA and MerB enzymes together increase the mercury metabolism spectrum in bacteria, improving bioremediation process. Check how the enzymatic pathway works on “Structure and mechanism” and it’s results in “Usage, Methodology and Experiments”.

Structure and mechanism:

This part is constituted by MerA, MerB, 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.

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!

To continue our characterization, we used this part in an improved Mer Operon, with new strongers regulated promoters, to increase mercury bioremediation, as you can see in the synthetic genetic circuits below.

We used two new regulated promoters (BBa_) to compare with natural one. The first test was validated the growth curve in 7.5ppm of HgCl2 in liquid LM media. The result are shown below.

As we can see, the two ones with the greater performance was the improved one, almost 4,6 times better than the previous device. It can be explain by its stronger promoter which increased the mer operon expression, turning bacteria more resistant to mercury! We used the best construction and measured the amount of mercury bioremediated, utilizing DMA-80 (Direct Mercury Analyzer).

The curve from the new construction reached 97% of mercury bioremediation showing its potential to depollute contaminated waters. So, to validate it, our team constructed the first real bioreactor for mercury bioremediation of iGEM! See the results below!

After 18h, our construction reached 70% of mercury bioremediation! Want to see more? Access our wiki: 2016.igem.org/Team:UFAM-UEA_Brazil.

Sequence and Features