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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.

2. Qualitative assay of attenuator

We try to check the attenuator decrease transcription with antisense. To confirm whether the attenuator-GFP express or not, we’ll induce expression of antisense RNA by IPTG on plate. As negative controls, we’ll use RNA with antisense-no IPTG, no-antisense and IPTG, spinach, and with tetRaptamer. As positive controls, we use RNA with Pcon-attenuator-GFP. As a result, we’ll expect the colony with antisense express the white for suppression GFP. The other hand, the others as negative and positive controls express the blue for GFP.

3. Qualitative assay of spinach 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.

4. Qualitative assay of tetRaptamer We’ll determine the quantity how far the guidance control of Ptet change by the expression of tetRaptamer. We’ll cultivate RNA on a liquid culture with IPTG, we’ll pick out a part at even intervals and determine the quantity both of it and the downstream ftom tetRaptamer and Ptet by using qRT-PCR. As negative controls, we won’t insert the promoter upstream from the downstream genes. As positive controls, using Pcon will express the downstream genes. To assay positive feedback, we’ll experiment the RNA with Ptet-Plac-(attenuator)-tetRaptamer. We think that we use qRT-PCR twice, one time when there are some time after inducing and when the change of transcription is bigger. As a result, we expect positive correlation between the quantity of tetRaptamer and the downstream genes. Considering the character of tetRaptamer, we will see the qualntity of downstream genes transcription start to induce after a part quantity of tetRaptamer transcription. About positive feedback circuit, Ptet-induction will start to induce after tetRaptamer' induction IPTG , so we think this change.

5. quantitively assay attenuator We check the amount of RNA transcribed from the gene downstream of attenuator region to measure the extent to which antisense repress the transcription by q-RT-PCR.We use two kind of E.coli:One brings Plac-antisense-spinach and Pcon-attenuator-tetR aptamer gene and one do not. We induce transcription by adding IPTG in liquid culture. We measure the quantity of mRNA in some periods from the start point of inducing to the time when transcription is comletely repressed. The amount of tetR aptamer will decease following the decrease of the amount of antisense. Comparing 4 experiment the change of antisense repression will be more gradual.

6. Cascade Assay tetRAptamer-Atntisense-Attenuator In order to realize transcriptional regulation system concerned with two kinds of functional RNA, we will try to compose system consist of TetR aptamer, attenuator, and antisense. We will introduce TetR aptamer at the downstream of Ptet and Plac, antisense combined with spinarch controlled by Ptet, attenuator-lacZ at the downstream of Pcon, and tetR controlled by Pcon into E.coli. As positive control, we will replace attenuator-lacZ with lacZ. As negative control, We will remove attenuator-lacZ.

7. Cascade Assay Antisense-Attenuator-tetRaptamer

Through using Antisese to repress tetR aptamer, we wii confirm the operation of cascade which repress the perform of lacZ.

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. 1,Due to qualitative, we will culture the liliquid with/without IPTG until the induction, and add X-gal.
2,Due to quantitative determination,after culturing the liquid with/without IPTG, in the more early point, we will measure some point of antisense, tetR aptamer, and lacZ by qRT-PCR.

8,Confirming Oscillation


Confirm oscillation at Final Construction


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 expecting that the oscillation period will be 10 minutes, so it would be enough to observe 1 hour. We are going to borrow fluorescence microscope of Prof. Matsuda, who belongs to graduate school of bio study.

In order to check whether this system works well, for qualitative assay, we will perform β-gal assay. We will incubate with IPTG for a while, and we will add X-gal. If this system works well, cells will become blue with IPTG and still white without IPTG. In addition, for quantitative assay, after E.coli increase sufficiently, we will add IPTG and we will perform qRT-PCR for the 3 kinds of functional RNAs a few times.We are expecting that most transcribed RNA will change according to time change in following order: Attenuator -> tetR aptamer, attenuator -> tetR aptamer, antisense, attenuator -> tetR aptamer, antisense