Kyoto:projectRNA/futureview

FutureView-assay

Assay

1. Qualitative assay of tetR

inducible tetR aptamer to drive GFP-expression on plate. As negative controls, we will use RNA with antisense, attenuator, spinach, no-RNA and attenuator-tetR aptamer. As positive controls, we will also use GFP. As a consequence, we will expect the colony analyzing X-gal express the blue with &beta-gal, the negative control express the white, and that the positive control do the blue.

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3. Qualitative assay of spinach assay

We will check that DFHBI fluorescence on a plate with spinach. We cultivate IPTG-inducible Spinach in a liquid culture under a shading condition, and add DFHBI. Then we check whether this sample fluorescence after centrifugation. We also check spinach-GFP and antisense-spinach.As a result, we expect the pellet fluorescence just with spinach and DFHBI.

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7. Purpose Through using Antisese to repress tetR aptamer, we wii confirm the operation of cascade which repress the perform of lacZ. Method Using Plac-antisense-spinach, Pcon-attenuator-tetR aptamer and Ptet-lacZ, we will compare it with IPTG introduction or without IPTG introduction. We have the control without Pcon-lacZ, attenuator and Ptet.

8,Confirming Oscillation

Aim Confirm oscillation at Final Construction Method We will enter Final Construction in cells to start oscillation by IPTG induction. We will culture the cells in liquid medium with IPTG, put a part of medium into observing solution with DFHBI in a plate, and observe 1 cell by fluorescence microscope at all time. Record it with CCD camera. Observing condition is not fixed yet. There are some previous researches about a cell oscillation, so we will decide on our policy referring to them and also essay of spinach. We are