Part:BBa_K2123201
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:
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
- 10COMPATIBLE WITH RFC[10]
- 12INCOMPATIBLE WITH RFC[12]Illegal NheI site found at 458
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal NgoMIV site found at 56
Illegal NgoMIV site found at 630 - 1000INCOMPATIBLE WITH RFC[1000]Illegal SapI site found at 49
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