Difference between revisions of "Part:BBa K1614016:Experience"

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Applications of BBa_K1614016

Material and Methods

Malachite Green Aptamer activity was tested by transcribing the prepared DNA-Template in vitro with T7 polymerase as described above and in presence of 1 mM malachite green (Sigma). To ensure synchronic initiation a master mix containing the buffer, enzymes, dye and template were pipetted separately. The assay was performed in 384 well micro titer plates (black, flat round, transparent bottom [Corning, 3540]) on a 20 µL scale. Evaporation during transcription was prevented by using a sealing tape. Measurements on micro titer plates were performed in a microplate reader (Tecan Safire 2). Following parameters were chosen for the assay setup: Ex= 630 nm and Em=652 nm, 10 nm excitation/ emission bandwidth, high sensitivity flash mode and 40 µs integration time. The reaction was measured every 30 sec at 37 °C. To ensure the functionality of the assay, samples of the in vitro transcription were analyzed on a 10 % 8 M urea PAGE.

Results

The results have shown that the fluorescence of the designed Malachite Green is dependend on the malachite green dye. Under conditions containing DNA, T7 RNA Polymerase and malachite green dye, we were able to observe an increase of fluorescence during in vitro transcription(Fig. 1).

Fig.1. Real time monitoring of the RNA synthesis using a Malachite Green Aptamer.(A) To analyze RNA synthesis in real time we applied a DNA template encoding for the RNA of interest (ROI) and a hammerhead ribozyme (HHR). Both fragments are inserted between the promotor (T7) and the Malachite Green Aptamer (MGA). Inducing the hammerhead ribozyme allows cleavage during the in vitrotranscription. Hereby two RNA fragments emerge, the ROI and the hammerhead ribozyme fused to Malachite Green Aptamer (HHR-MGA). Using such setup, the emitted fluorescence of the HHR-MGA will be independent on the applied ROI. Thus the fluorescence is induced by the Malachite Green Aptamer that can be applied to compare efficiencies of different RNAs at the same time. (B) As a proof of principle we performed transcriptions and monitored the malachite green fluorescence signal in real time. (C)To confirm the results of the fluorescence measurements as well as the transcription efficiency is not hampered by the malachite green dye, all in vitro transcription reactions were analyzed using denaturing acrylamide gels.

User Reviews

UNIQee7c528bff2a02fa-partinfo-00000000-QINU UNIQee7c528bff2a02fa-partinfo-00000001-QINU

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 ===Applications of BBa_K1614016===
-Establishing a read out system that would give the information that the DNA was fully transcribed into RNA was provided by the Malachite Green Aptamer. Therefore we provide a new biobrick which contains a hammerhead ribozyme fused to the Malachite Green Aptamer (BBa_K1614016). This aptamer can bind malachite green dye which lead to a fluorescence at 652 nm if excited at 630 nm. This part was used for the detection of fully extended RNA during <i> in vitro </i> transcription in presence of malachite green dye. The ligand dependent aptamer folds correctly during the transcription thereby emitting light that was measured in a 384 well plate format. Experiments have shown that the Malachite Green Aptamer can be used as a universal read out system by adding a Hammerhead Ribozyme that cleaves at the 3’ end of the RNA of Interest. This setup allows the cleavage of an RNA of Interest (ROI) from the Hammerhead Ribozyme-Malachite Green Aptamer.
+==Material and Methods==
-Experiments with the Substrate of the RNA cleaving DNAzyme have shown that the Hammerhead Ribozyme-Malachite Green Aptamer cleaves from the substrate. Hence, the substrate and the Hammerhead Ribozyme-Malachite Green Aptamer could be purified on a denaturing polyacrylamide gel electrophoresis (Fig. 1).
+Malachite Green Aptamer activity was tested by transcribing the prepared DNA-Template <i>in vitro</i> with T7 polymerase as described above and in presence of 1 mM  malachite green (Sigma). To ensure synchronic initiation a master mix containing the buffer, enzymes, dye and template were pipetted separately. The assay was performed in 384 well micro titer plates (black, flat round, transparent bottom [Corning, 3540]) on a 20 µL scale. Evaporation during transcription was prevented by using a sealing tape. Measurements on micro titer plates were performed in a microplate reader (Tecan Safire 2). Following parameters were chosen for the assay setup: Ex= 630 nm and Em=652 nm, 10 nm excitation/ emission bandwidth, high sensitivity flash mode and 40 µs integration time. The reaction was measured every 30 sec at 37 °C. To ensure the functionality of the assay, samples of the <i>in vitro</i> transcription were analyzed on a 10 % 8 M urea PAGE.
-[[File:Figure_6_final.png|thumb|700 px|center|'''Fig.1. Real time monitoring of the RNA synthesis using a Malachite Green Aptamer.(A)''' To analyze RNA synthesis in real time we applied a DNA template encoding for the RNA of interest (ROI) and a hammerhead ribozyme (HHR). Both fragments are inserted between the promotor (T7) and the Malachite Green Aptamer (MGA). Inducing the hammerhead ribozyme allows cleavage during the in vitrotranscription. Hereby two RNA fragments emerge, the ROI and the hammerhead ribozyme fused to Malachite Green Aptamer (HHR-MGA). Using such setup, the emitted fluorescence of the HHR-MGA will be independent on the applied ROI. Thus the fluorescence is induced by the Malachite Green Aptamer that can be applied to compare efficiencies of different RNAs at the same time. '''(B)''' As a proof of principle we performed transcriptions and monitored the malachite green fluorescence signal in real time. '''(C)'''To confirm the results of the fluorescence measurements as well as the transcription efficiency is not hampered by the malachite green dye, all in vitrotranscription reactions were analyzed using denaturing acrylamide gels.]]
+==Results==
+The results have shown that the fluorescence of the designed Malachite Green is dependend on the malachite green dye. Under conditions containing DNA, T7 RNA Polymerase and malachite green dye, we were able to observe an increase of fluorescence during <i> in vitro </i> transcription(Fig. 1).
+[[File:Figure_6_final.png|thumb|700 px|center|'''Fig.1. Real time monitoring of the RNA synthesis using a Malachite Green Aptamer.(A)''' To analyze RNA synthesis in real time we applied a DNA template encoding for the RNA of interest (ROI) and a hammerhead ribozyme (HHR). Both fragments are inserted between the promotor (T7) and the Malachite Green Aptamer (MGA). Inducing the hammerhead ribozyme allows cleavage during the in vitrotranscription. Hereby two RNA fragments emerge, the ROI and the hammerhead ribozyme fused to Malachite Green Aptamer (HHR-MGA). Using such setup, the emitted fluorescence of the HHR-MGA will be independent on the applied ROI. Thus the fluorescence is induced by the Malachite Green Aptamer that can be applied to compare efficiencies of different RNAs at the same time. '''(B)''' As a proof of principle we performed transcriptions and monitored the malachite green fluorescence signal in real time. '''(C)'''To confirm the results of the fluorescence measurements as well as the transcription efficiency is not hampered by the malachite green dye, all in vitro transcription reactions were analyzed using denaturing acrylamide gels.]]
 ===User Reviews===