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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.

PSB1A3_RCNA+ YFP:

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 than normal individuals.

To prove that our construct works, we made some tests with transformed E. coli XL1-Blue. We added different concentrations of cobaltous chloride to bacterial cultures and measured their fluorescence (excitation: 514nm; emission: 527 nm) and absorbance (600 nm) for a certain period. The results for fluorescence(Figures 6) show a peak of fluorescence which can be seen 1 hour after 25 µM treatment, and another peak after 4 hours of 75µM treatment. However,normalizing the data according absorbance measures we can see only a peak after 4 hours of 75µM treatment.

Figure 6: 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. A peak of fluorescence can be seen 3 hours after treatment. Intermediate concentrations of cobalt were more efficient in generating fluorescence.

Figure 8: Fluorimetric and absorbance reads of cultures of E. coli XL1-Blue carrying the plasmid PSB1A3_RCNA+ YFP, after treatment with different concentrations of cobalt. The fluorescence reads shown in figures 6 and 7 were divided by the absorbance, resulting in the graphic above. The 75 µM concentration of cobalt was more efficient in generating fluorescence 4 hours after treatment.

As IMA binds less to cobalt than normal albumin, we expected that a serum containing more IMA 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 a patient with cardiac risk from a normal patient by comparing the fluorescence generate by each serum.

First, we tested whether BSA (bovine serum albumin)would produce the expected result (more BSA, less free cobalt, less fluorescence). As can be seen on Figure 9, the result obtained meets the expected result.

Figure 9: Fluorimetric assay to assess the BSA cobalt binding. In this experiment we measured the fluorescence produced by the RCNA-YFP modified E.coli according to the quantity of Bovine Serum Albumin (BSA) and 100 µM of cobalt in the media. Each bar shows the percentual fluorescence of each BSA concentration tested according our positive control (culture + 100 µM of cobalt). The fluorescence was measured minutes after treatment. As we can see, the fluorescence produced by bacteria is higher for less concentrations of BSA.

We have also added mice serum to bacteria containing the composite. These sera were from ischemic or non ischemic mice.

We can see that for two of the ischemic serum samples the fluorescences produced were higher during the entire experiment, and we can differentiate it from the normal serum samples. However, for one ischemic serum sample, its fluorescence pattern can be differentiated from the normal samples just at the zero time. We hypothesized that it happened maybe because its degree of ischemia is smaller than the others (Figure 10).

Figure 10: Fluorimetric assay IMA versus non IMA cobalt binding. In this experiment was measured how much cobalt is free in the mice serum by using RCNA-YFP modified E. coli according to the quantity of cobalt in the serum. We used two different samples of mice, each one in triplicate: ischemic and non-ischemic serum. The three curves more above (Isq1, Isq2, Isq3) are the serums with ischemic-albumin and the the two below (Normal 1, Normal 2), the non ischemic.

PSB1C3_TorCAD+RFP:

We have constructed the composite TorCAD + RFP 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 a certain period. The results (Figures 11 to 12) show a peak after 3 hours, during exponential phase.

Figure 11: Fluorimetric 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 12: Fluorimetric 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 maximum of fluorescence reached in the E. coli XL1-Blue carrying the plasmid PSB1A3_RCNA+ YFP occurred 4 hours after the treatment with 75µM of cobalt, as showed in the figure 8. Even before eight hours after the treatment we can see that the fluorescence is still higher than the other concentrations of test.

It is noteworthy the importance of data normalization according the absorbance measure, because different amounts of bacteria will result in different fluorescence values (Figure 6), but when we see the measure of fluorescence per measure of absorbance we conclude that the best concentration of cobalt for the sensor activation is 75 µM of cobalt (Figure 8).

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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