Part:BBa_K1086001:Experience

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Applications of BBa_K1086001

We have constructed the composite RCNA+YFP to detect IMA (ischemia modified albumin) in the serum of patients with cardiac risk. These patients present more IMA, consequently less normal albumin than normal individuals.

To prove that our construct works, we transformed E. coli XL1-Blue and tested different concentrations of cobaltous chloride in bacterial cultures measuring their fluorescence (excitation: 514nm; emission: 527 nm) and absorbance (600 nm) for a certain period.

The fluorometric essay (Figures 1) show a peak of fluorescence in 3 hours after the treatment with the different cobaltous chloride concentration (0, 25, 50, 75, 100, 125 or 150 µM). The higher fluorescence intensity could be observed with 100 and 125 µM of cobaltous chloride.

Figure 1: Fluorometric reads of cultures of E. coli XL1-Blue carrying the plasmid PSB1A3_RCNA+ YFP, along the time, after treatment with 0, 25, 50, 75, 100, 125 or 150 µM of cobaltous chloride.

With both absorbance and fluorescence, the normalized signal (Figure 2) showed the higher fluorescence by absorbance with the concentration of 100 µM of cobaltous chloride 3 hours after treatment. The inobservance of signal with 125 or 150 µM of cobaltous chloride could be caused by toxicity of these concentrations for bacteria.

Figure 2: Fluorometric and absorbance reads of cultures of E. coli XL1-Blue carrying the plasmid PSB1A3_RCNA+ YFP, after treatment with different concentrations of cobaltous chloride. Bacteria were treated with 0, 25, 50, 75, 100, 125 or 150 µM of cobaltous chloride. After that, fluorescence and absorbance were read hourly, until 4 hours, and there were read 8 and 24 hours after treatment.

We further tested the cobalt binding capacity of sera from isquemic and non-isquemic mice using the modified E. coli. As IMA binds less to cobalt than normal albumin, we expected that a serum containing more IMA (and less normal albumin- isquemic mice) would have more free cobalt than a “normal” serum. This excess of cobalt would be able to activate more RCNA promoter, which in turn would lead to expression of YFP, so we could be able to distinguish between isquemic and normal mice comparing the fluorescence generate by each serum, exactly the results observed in our experiment (Figure 3).

Figure 3: Fluorometric assay using serum from isquemic and non-isquemic (normal) mice. In this experiment, 100 μM of cobaltous chloride was incubated with serum from 3 different mices with induced isquemia or not using RCNA-YFP modified E. coli. The three curves above (Isq1, Isq2, Isq3) are the sera of ischemic mice and the three below (Nor1, Nor2, Nor3), the non ischemic mice sera.

Conclusion:

Our results show that the composite RCNA+YFP generates fluorescence in the presence of cobalt in different concentrations. Furthermore, it can be used to distinguish between ischemic and non ischemic individuals.

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