Difference between revisions of "Part:BBa K2123201"

Latest revision as of 03:50, 28 October 2016

Strong RBS + MerB (Organomercurial Lyase) + Strong RBS + MerA (Mercuric Reductase) + B0015

Overview

This composite part was developed to turn available two mer operon enzymes, related to Hg metabolism: organomercurial lyase (MerB) and mercury reductase (MerA), as you can see below.

With this part, you can switch: I) promoter sequences, regulating (or not) by what you want; II) transporters protein, using the preferential one for your chassis; III) and whatever you want related to mercury metabolism! These two 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:

Mercuric ion reductase, codified by MerA, presents itself as an homodimer. It has two terminal sites, each containing a pair of cysteines: a short and mobile C-terminal and a long N-terminal. At the active site, four cysteine residues engage in Hg(II) binding, but not take part in reduction. C-terminal cysteines are responsible for catching Hg(II) ions from solution and delivering them to the active site, where the pair of inner cysteines would establish chemical bonds with mercury. C-terminal cysteines are responsible for catching Hg(II) ions from solution and delivering them to the active site, where the pair of inner cysteines would establish chemical bonds with mercury. At the core site, FAD mediates the electron transfer between NADPH and Hg(II); primarily NADPH is oxidized to NADP+ while FAD is reduced to FADH-, then FADH- reduces ionic mercury Hg(II) to Hg0.

Organomercurial lyase, encoded by MerB, breaks the bond between Hg and a carbon atom from an organic radical, releasing Hg(II) to be reduced by MerA. In this complex, mercury has the ability to bind itself to biological tissues and propagate through food chain until it reaches humans, a phenomenon called biomagnifications. The purified protein required a minimum two-fold molar excess of thiol over organomercurial substrate to exhibit any activity and preferred the physiological thiol cysteine to non-physiological mercaptans. The enzyme has a very broad substrate tolerance, handling both alkyl and aryl mercurials, with a slight preference for the latter. As is often the case with enzymes of low substrate specificity, MerB has very slow turnover rates ranging from 0.7 to 240 min−1 on various substrates. Although relatively slow for an enzyme, these rates do represent a 106–107-fold acceleration over chemical protonolysis rates of organomercurials.

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