Difference between revisions of "Part:BBa K1086002:Experience"

Latest revision as of 01:08, 29 October 2013

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Applications of BBa_K1086002

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.

TMAO enters bacterial cells and generates a cascade of signals, which ultimately activate promoter TorCAD (we have based on Ansaldi et al., 2010 to synthesize TorCAD promoter). We have added mCherry (RFP) downstream of TorCAD in order to measure the promoter’s activation by TMAO.

We made some experiments with E. coli XL1-Blue transformed with this composite to show that our construct works. We added different concentrations of TMAO and measured bacterial cultures fluorescence (excitation: 587 nm; emission: 610 nm) and absorbance (600 nm) for a certain period.

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 1) 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 1: 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.

Conclusion:

Our results show that the composite TorCAD+RFP generates fluorescence in the presence of TMAO.

User Reviews

UNIQ15c6d08a7e268ed3-partinfo-00000000-QINU UNIQ15c6d08a7e268ed3-partinfo-00000001-QINU

@@ Line 10: / Line 10: @@
 TMAO enters bacterial cells and generates a cascade of signals, which ultimately activate promoter TorCAD (we have based on Ansaldi et al., 2010 to synthesize TorCAD promoter). We have added mCherry (RFP) downstream of TorCAD in order to measure the promoter’s activation by TMAO.
-We made some experiments with ''E. coli'' XL1-Blue transformed with this composite to show that our construct works. We added different concentrations of TMAO and measured bacterial cultures fluorescence (excitation: 567 nm; emission: 610 nm) and absorbance (600 nm) for a certain period. The results (Figure 1) show a peak after 3 hours, during exponential phase.
+We made some experiments with ''E. coli'' XL1-Blue transformed with this composite to show that our construct works. We added different concentrations of TMAO and measured bacterial cultures fluorescence (excitation: 587 nm; emission: 610 nm) and absorbance (600 nm) for a certain period.
-[[File:Figure1-TMAO.jpg]]
+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 1) 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.jpg|600px|thumb|center|'''Figure 1: 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.]]