Composite

Part:BBa_K1355000

Designed by: Maria Clara Tavares Astolfi, Luna Barroco de Lacerda   Group: iGEM14_UFAM_BRAZIL   (2014-10-06)

Strong RBS + merA (mercuric ion reductase)+ terminator (BBa_ B0015)

The MerA gene is one of the most important gene into bacterial mercury-resistance operon. The mercuric reductase is a cytosolic flavin disulfide oxidoreductase (homodimer ∼120 kDa) which uses NAD(P)H as a reductant. Transforms mercury Hg2+ in Hg0, which volatile, lipid-soluble Hg(0) diffuses through the cell membranes without the need for any dedicated efflux system, as shown in figure below.


L1.png

Figure 01: Mer genes action in bacterial cell

BacMERCURY.jpg

Source

MerA gene sequence is found in the O26-CRL plasmid from Escherichia coli O26. We also added strong RBS from protein 10 found in phage 17 and the double terminator (BBa_B0015) to ensure efficient termination of transcription and messenger RNA recognition. P026-crl.jpg


Experiments and Results as a BIOREMEDIATOR

BBa_K1355001 (bidirectional promoter regulated by the MerR) + BBa_K1355000

The experiment for Hg bioremediation was made according to the protocol “Cell growth quantification of mercury resistant Escherichia coli DH5α at differents Hg concentrations by Optical Density”. DH5-alpha transformed with BBa_K1355004 was inoculated in LM (LB with low concentration of NaCl) liquid medium with 0.05μg/ml of mercury chloride and 34μg/ml of chloramphenicol. The inoculum was incubated in shaker overnight at 37°C. After cell growth, we measured Optical Density (O.D.) of the cell culture in spectrophotometer at 600 nm wavelength. A standard quantity aliquot of bacterial suspension was taken in six falcons (50ml) with 10 ml of LM liquid medium and then added mercury chloride in order to achieve the concentrations: 0μg/ml, 1 μg/ml; 2.5 μg/ml; 5 μg/ml; 10μg/ml; 20 μg/ml. The samples were incubated at 37°C on shaker. We collected each sample at the given times, 1 (02:30 hours of incubation), time 2 (04:30 hours of incubation), time 3 (06:30 hours of incubation), time 4 (17 hours of incubation), time 5 (24 hours of incubation) to measure O.D. on spectrophotometer. As control, we used DH5-alpha transformed with BBa_K1355002 (Hg bio detector device) which does not present coding regions for the mercuric ion reductase. We also measured Optical Density of each sample. After cell growth measurement, the samples were frozen to subsequently quantification of Hg remediated by the Mercury Bacter.

The graph represented on Figure 1 and Figure 2 shows Cell growth of Mercury Bacter and of the control bacteria in different Hg concentrations in function of time, respectively;

Br1.png

Figure 1: Cell growth of Mercury Bacter in different Hg concentrations in function of time;

Br2.png

Figure 2: Cell growth of control bacteria in different Hg concentrations in function of time

Comparing the graphs shown on figure 1 and 2, it can be observed that the biobrick BBa_K135504 provided resistance to high Hg concentrations. Control bacteria did not present resistance to Hg concentration equal or higher to 1 ppm. On the contrary, Mercury Bacter grew up even in 10 ppm Hg concentrations. Interestingly, at the concentration of 10 ppm Mercury Bacter needed 24 hours to exhibit increase in cell growth, and after 48 hours it maintained the same OD, unlike the others. At 1 ppm, 2.5 ppm and 5 ppm Mercury Bacter performed an overwhelming cellular growth after 24 hours of exposure to Hg, reaching approximately the same cellular growth as the non-exposed bacteria!!

The graph represented on Figure 3 compares Hg concentration in LM medium after 48 hours of experiment, between medium with bacteria used as control and engineered Mercury Bacter.

Br3.png

Figure 3: Hg concentration in LM medium after 48 hours of cell growth in control bacteria and in the genetically engineered Mercury Bacter.

In the graph above it can be observed that our super Mercury Bacter bioremediated! In action, Mercury Bacter reduced 72% of the mercury levels, in comparison to control at the sample 5 ppm! In 1 ppm, Even though on 10 ppm concentration, where bacteria had a slow growth rate only after 24 hours, it bioremediated 48.02%! These results demonstrate our constructions’ efficiency, both for biobrick BBa_K1355001 as a promoter regulated by MerR protein and to BBa_K1355000 as MerA encoding gene!


References

Barkay, T., Miller, S. M., & Summers, A. O. (2003). Bacterial mercury resistance from atoms to ecosystems. FEMS microbiology reviews, 27(2‐3), 355-384.

BIONDO, R. Engenharia Genética de Cupriavidus metallidurans CH34 para a Biorremediação de efluentes contendo Metais Pesados. 2008. São Paulo, Brasil.

Nascimento, A. M., & Chartone-Souza, E. (2003). Operon mer: bacterial resistance to mercury and potential for bioremediation of contaminated environments. Genetics and Molecular Research, 2(1), 92-101.

DASH, H. R.; DAS, S. Bioremediation of mercury and the importance of bacterial mer genes. 2012. International Biodeterioration & Biodegradation 75: 207 – 213.

HAMLETT, N. V.; et al. Roles of the Tn21 merT, merP and merC Gene Products in Mercury Resistance and Mercury Binding. 1992. Journal of Bacteriology 174: 6377 – 6385.

PINTO, M. N. Bases Moleculares da resistência ao Mercúrio em bactérias gram-negativas da Amazônia brasileira. 2004. Pará, Brasil.

Sequence and Features

Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal NgoMIV site found at 1200
    Illegal NgoMIV site found at 1262
  • 1000
    COMPATIBLE WITH RFC[1000]


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Categories
//binding/metal
Parameters
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