Introduction
A biosensor is a system composed of a biological transducing element that produces measurable signal in response to environmental changes. A good example of biosensor, are the bioreporters bacteria that produce visible light in answer to chemicals. Allowing the detection of toxic elements.
The use of genetically modified bacteria was a big step for synthetic biology and became popular for its benefits, including detection in soil and water for heavy metal pollution. In order to have a bioreporter able to be used under different concentrations of mercury, we built a biosensor using Escherichia coli DH5-alpha which express the green fluorescent protein (GFP) only in the presence of this toxic element.
Why Green Fluorescent Protein?
Green fluorescent protein (GFP) cloned from Aequorea victoria, is a popular bioreporter, especially to observe gene expression in eukaryotic and bacteria cell. GFP is a stable protein, and it is autofluorescent, remaining fluorescent even after cell death. It doesn’t require a substrate, however, it needs oxygen and an energy donor to emit fluorescence. The toxic effects of metal concentration on fluorescent protein is less noticeable comparing to others bioreporters which make them better for rapid applications.
Our construction (How it works?)
To develop a biosensor in Escherichia coli DH5-alpha, we designed a biobrick device to express Green Fluorescent Protein in mercury’s occurrence. The Mercury ions’ detector device biobrick (BBa_K1355002) is composed by mer bidirectional promoter (BBa_K1355001) attached to the GFP translational unit (BBa_E0840). It has dual function: A) In reverse: MerR protein regulator transcription; and B) in forward: transcription of MerP - MerT - GFP proteins, as represented below:
In absence of mercury, MerR forms a MerR-promoter-operator complex, preventing RNA polymerase to recognize the promoter, consequently, messengers RNA for MerPT and GFP will not be transcript. In presence of Hg2+, MerR protein binds to this element and dissociates from the promoter-operator complex, allowing MerPT and GFP expression, as represented below:
Experiments and Results
The experiment to quantify GFP expression induced by Hg was made according to the protocol “Quantification of Green Fluorescent Protein (GFP) induced by different concentrations of mercury in Escherichia coli DH5α”.
DH5α transformed with BBa_K1355002 was inoculated in LM (LB with low concentration of NaCl) liquid medium with chloramphenicol and grew until the Optical Density was 0.4 to 0.6abs (measured on spectrophotometer at 600 nm wavelength). After cell growth, an aliquot of 500μl in 5 eppendorf tubes (2ml) was taken and then added mercury chloride in order to achieve the concentrations of: 0.01 µg/ml, 0.02 µg/ml, 0.1 µg/ml, 0.2 µg/ml, and 1 µg/ml. The samples were incubated at 37°C on shaker. We collected each eppendorf tube at time 1 (01:30 hours of incubation), time 2 (03:00 hours of incubation) and time 3 (04:30 hours of incubation). Every sample was centrifuged at 12000g for 3 minutes and the pellet washed with TN Buffer (Nacl 0.15M + Tris HCl 10mM) and then re-suspended with 500μl of the same buffer. The same process was made to the bacterium without construction as a control to GFP expression/intensity. GFP expression was measured using the Hidex Chameleon spectrofluorimeter with excitation filter 340 nm and emission filter 500 nm wavelength. The Optical Density was measured simultaneously. All samples were analyzed in triplicate.
The graph represented on Figure 1 shows the Optical Density of transformed DH5α with BBa_K1355002 in different Hg concentrations in function of time:
Figure 1. Optical Density measured in the four given times, at mercury chloride concentrations of 0 µg/ml, 0.01 µg/ml, 0.02 µg/ml, 0.1 µg/ml, 0.2 µg/ml, and 1 µg/ml.
In these condition cell growth increases along time. The highest values correspond to bacteria not exposed to mercury or to small concentrations, as in 0 µg/ml, 0.01 µg/ml and 0.02 µg/ml. Suggesting a harmless condition to bacteria. However, cell growth decreases at higher concentrations as, 0.1 µg/ml, 0.2 µg/ml and especially 1µg/ml, giving to bacteria a hard time for development.
The graph represented on Figure 2 shows the fluorescence emitted by DH5-alpha induced by different Hg concentrations in function of the time; and the graph represented on Figure 3, shows the ratio between fluorescence emitted and Optical Density.
Figure 2. GFP fluorescence intensity in the four given times, at mercury chloride concentrations of 0 µg/ml, 0.01 µg/ml, 0.02 µg/ml, 0.1 µg/ml, 0.2 µg/ml, and 1 µg/ml.
Reference
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