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Results

Constructs

PSB1A3_RCNA+ YFP:

Figure 1: Transformation of XL1-Blue bacteria with the ligation PSB1A3_RCNA+ YFP. Many bacteria grew on plates! We took some colonies and had their plasmids minipreped.

Figure 2: Digestion of PSB1A3_RCNA+ YFP miniprep. Digestions were made using EcoRI and PstI enzymes. A fragment of the expected size for RCNA+YFP (~ 1300 pb) can be seen in lane 4. 1Kb: molecular ladder. 1: non digested plasmid. 2: plasmid digested with EcoRI. 3: plasmid digested with PstI. 4: plasmid digested with EcoRI and PstI.

Figure 3: PCR of PSB1A3_RCNA+ YFP minipreps. PCRs were made using VF2 and VR primers. A fragment of the expected size for RCNA+YFP (~ 1300 pb) can be seen in lanes 1 and 3. 1Kb: molecular ladder. 1: miniprep 1. 2: miniprep 2. 3: miniprep 3. 4: miniprep 4. C-: negative control.

PSB1C3_TorCAD:

Figure 4: PCR of PSB1A3_TorCAD minipreps. PCRs were made using VF2 and VR primers. Fragments of the expected size for TorCAD (~ 120 pb) can be seen in all lanes. 1Kb: molecular ladder. 1: miniprep 1. 2: miniprep 2. 3: miniprep 3. 4: miniprep 4.

PSB1C3_TorCAD+RFP:

Figure 5: PCR of PSB1A3_TorCAD+RFP colonies. PCRs were made using VF2 and VR primers. Fragments of the expected size for TorCAD+RFP (~ 1000 pb) can be seen in some lanes. 1Kb: molecular ladder. 1 to 10: 10 different colonies used as templates for PCR.

Fluorometric Assays

The results shown in here were performed as described in Protocols. We used Varioskan Flash Multimode Reader (Thermo Scientific™) to do the reads of YFP constructs and Synergy 2 (Biotek) to do the reads of RFP constructs.

PSB1A3_RCNA+ YFP:

We have constructed the composite RCNA+YFP BBa_K1086001 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 6) 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 6: 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 7) 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 7: 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.

Further, we tested whether BSA (bovine serum albumin) in different concentration would produce the expected result (more BSA, less free cobalt, less fluorescence). As can be seen on Figure 8, the result obtained meets the expected result, where the BSA concentration of 66 mg/ml were not able to generate a fluorescence signal different from the smaller BSA concentration of 0.002 mg/ml.

Figure 8: Fluorometric assay to assess the BSA cobalt binding. In this experiment we measured the fluorescence produced by the RCNA-YFP modified E.coli incubated with 100 μM of cobaltous chloride and different Bovine Serum Albumin (BSA) concentrations. Each curve shows different concentrations of Bovine Serum Albumin (BSA) and its respective fluorescence along the time. As we can see, the fluorescence produced by bacteria increases according to the cobalt available in the media.

We further tested the cobalt binding capacity of sera from isquemic and non-isquemic mice using the modified E. coli. The sera from animals were obtained as described in Protocols. 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 9).

Figure 9: 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. .

We can conclude that the three curves with most intense fluorescence are due to the ineffective cobalt chelation by the mice serum albumin. In the control sample we can see the opposite effect, showing our E. coli sensor working as expected. Great!!

PSB1C3_TorCAD+RFP:

We have constructed the composite TorCAD + RFP Ba_K1086002 to detect TMAO (Trimethylamine N-oxide) in the serum of patients with cardiac risk. These patients present more TMAO than normal individuals.

To prove that our construct works, we made some tests with transformed E. coli XL1-Blue. We added different concentrations of TMAO to bacterial cultures and measured their fluorescence and absorbance for 15 hours. The fluorescence stars to increase only 8 hours after the treatment with TMAO (Figures 10 and 11) and continue to increase until 13 hours after the treatment. We could observe fluorescence appearence only with the concentration of 100 μM of TMAO.

Figure 10: Fluorometric reads of cultures of E. coli XL1-Blue carrying the plasmid PSB1C3_TorCAD + RFP, after treatment with different concentrations of TMAO. Bacteria were treated with 0 μM, 1 μM, 10 μM, 100 μM, 1 mM, 10 mM and 100 mM. After that, fluorescence was read hourly, until 15 hours. The beginning of the fluorescence increase can be seen about seven hours after the treatment.

Figure 11: Fluorometric and absorbance reads of cultures of E. coli XL1-Blue carrying the plasmid PSB1C3_TorCAD + RFP, after treatment with different concentrations of TMAO. The fluorescence reads shown in figure 11 divided by the absorbance, resulting in the graphic above.

Discussion and Conclusions

The peak of fluorescence after 3 hours (Figures 6 to 8) is probably related to the phase of growth in which bacteria are (exponential phase). At this phase, bacteria are more metabolically active, once they are dividing in a great rate, so they need to produce large amounts of proteins.

Concerning cobalt concentrations, it is likely that lower concentrations activate the promoter less than intermediate concentrations, whereas bigger concentrations might be saturating the promoter, or even causing negative feedback.

In the tests using BSA or mice sera, the results meet our model, in which more normal albumin(or BSA) leads to less free cobalt, resulting in lower fluorescence.

Thus, our results show that the composite RCNA+YFP generates fluorescence in the presence of cobalt. Furthermore, it can be used to distinguish between ischemic and non ischemic individuals. Further characterization, including usage of samples containing human IMA (ischemia modified albumin) and normal albumin, is needed, in order to improve our composite’s documentation.

Regarding TMAO, we found that the fluorescence increases after 7 hours of 100 µM TMAO treatment, showing that our TorCAD+RFP worked as expected. Further characterization using human sera is also needed for better composite’s documentation.

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