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Latest revision as of 01:08, 29 October 2013

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 8 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.

Conclusions and Perspectives

During the time of the competition our team was able to:

Modify the previous described biobrick BBa_K540001 adding a fluorescent yellow protein and give it a new functionality. Our biobrick BBa_K1086001 is able to differentiate serum from isquemic and non isquemic mice.

Generate a new Biobrick BBa_K1086002 able to detect a concentration of 100 µM of TMAO.

Generate modeling that describe our systems and simulate it.

Expand the knowledge about synthetic biology in our University, our State and in Brazil.

As a perspective of this work, we aim:

To incorporate more plasmid copies with Tor_CAD BBa_K1086002 biobrick to decrease the time to detect TMAO and to detect less TMAO concentrations.

To test our 2 constructs with isquemic and non isquemic pacients serum.

To test our 2 constructs with serum from obese and non-obese patients to try to predict the potential risk of cardiovascular events in these patients.

Our Sponsors

@@ Line 49: / Line 49: @@
-The results shown in here were performed as described in <html><a href='https://2013.igem.org/Team:UFMG_Brazil/Protocols'>Protocols</a></html>. We used Varioskan Flash Multimode Reader (Thermo Scientific™) to do the reads.
+The results shown in here were performed as described in <html><a href='https://2013.igem.org/Team:UFMG_Brazil/Protocols'>Protocols</a></html>. 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.
@@ Line 55: / Line 55: @@
-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.
+We have constructed the composite RCNA+YFP <html><a href=http://parts.igem.org/cgi/partsdb/pgroup.cgi?pgroup=iGEM2013&group=UFMG_Brazil>BBa_K1086001</a></html> 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 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.
+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.
 <!--Isso não está sendo mostrado-->
-[[File:RCNAfluor.jpg|700px|thumb|center|]]
+[[File:RCNAfluor.jpg|700px|thumb|center|'''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.
-[[File:Ima fluo curva.jpg|700px|thumb|center|"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.]]
+[[File:RCNAFLUORABSO.jpg|700px|thumb|center|'''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.]]
-<!--[[File:ima_fluo_barra.jpg|700px|thumb|center|''Fluorometric reads of cultures of ''E. coli'' XL1-Blue carrying the plasmid PSB1A3_RCNA+ YFP, after treatment with different concentrations of cobalt.''' This result is the same as the one shown in figure 6, but here the focus is at the points where peaks appeared'.]]-->
+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.
-<!--[[File:Ima fluo curva.jpg|700px|thumb|center|'''Figure 6: Fluorimetric reads of cultures of ''E. coli'' XL1-Blue carrying the plasmid PSB1A3_RCNA+ YFP, after treatment with different concentrations of cobalt.''' This result is the same as the one shown in figure 6, but here the focus is at the points where peaks appeared.]]-->
+[[File:bsa_assay.jpg|500px|thumb|center|'''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 <html><a href='https://2013.igem.org/Team:UFMG_Brazil/Protocols'>Protocols</a></html>.
+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).
-<!--[[File:ima_fluo_per_abs.jpg|700px|thumb|center|'''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.]]-->
+[[File:normal_vs_isquemic.jpg|700px|thumb|center|'''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!!
-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.
+'''PSB1C3_TorCAD+RFP:'''
-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.
+We have constructed the composite TorCAD + RFP <html><a href=http://parts.igem.org/cgi/partsdb/pgroup.cgi?pgroup=iGEM2013&group=UFMG_Brazil>Ba_K1086002</a></html> 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.
+[[File:clone2_tmao_fluo.jpg|600px|thumb|center|'''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 8 hours after the treatment.]]
-[[File:bsa_assay.jpg|500px|thumb|center|'''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.]]
+[[File:clone2_tmao_fluo_per_abs.jpg|600px|thumb|center|'''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.]]
+== Conclusions and Perspectives ==
+During the time of the competition our team was able to:
-We have also added mice serum to bacteria containing the composite. These sera were from ischemic or non ischemic mice.
+* Modify the previous described biobrick <html><a href=http://parts.igem.org/Part:BBa_K540001>BBa_K540001</a></html> adding a fluorescent yellow protein and give it a new functionality. Our biobrick <html><a href=http://parts.igem.org/cgi/partsdb/pgroup.cgi?pgroup=iGEM2013&group=UFMG_Brazil>BBa_K1086001</a></html> is able to differentiate serum from isquemic and non isquemic 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).
-[[File:normal_vs_isquemic.jpg|700px|thumb|center|'''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.
-[[File:clone2_tmao_fluo.jpg|600px|thumb|center|'''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.]]
+* Generate a new Biobrick <html><a href=http://parts.igem.org/cgi/partsdb/pgroup.cgi?pgroup=iGEM2013&group=UFMG_Brazil>BBa_K1086002</a></html> able to detect a concentration  of 100 µM of TMAO.
-[[File:clone2_tmao_fluo_per_abs.jpg|600px|thumb|center|'''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.]]
+* Generate modeling that describe our systems and simulate it.
-== Discussion and Conclusions ==
+* Expand the knowledge about synthetic biology in our University, our State and in Brazil.
-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.
+As a perspective of this work, we aim:
-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).
+* To incorporate more plasmid copies with Tor_CAD <html><a href=http://parts.igem.org/cgi/partsdb/pgroup.cgi?pgroup=iGEM2013&group=UFMG_Brazil>BBa_K1086002</a></html> biobrick to decrease the time to detect TMAO and to detect less TMAO concentrations.
-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.
+* To test our 2 constructs with isquemic and non isquemic pacients serum.
-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.
+* To test our 2 constructs with serum from obese and non-obese patients to try to predict the potential risk of cardiovascular events in these patients.
-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.
 {{Team:UFMG Brazil/sponsor}}

Team:UFMG Brazil/Results