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Basic Flip Flop. Results

The most important features of the basic bistable are the following:

• Time of the change of state

• Stability when the inducer is retired

So these are the ones that I have studied more accurately in my experiments. I set overnight inoculums inducing a condition (adding 0,1 mM IPTG or growing at 42ºC). The morning after I make a 1/100 dilution, and let it grow until de optic density reaches 0,2. In that moment I change the conditions, to test the change between states or just retire the inducers, to observe the stability of one state in a long time.

We made the experiments in two ways:

• Qualitative, assessing that the changes actually occur. For this, we use the fluorescence microscope.

• Quantitative, with the microtiter reader we can measure how big the changes are and how quick they happen.

It’s important not to use LB media, because it contains cyclic aminoacids which make it fluorescent. For all this experiments we used Minimal Media supplemented with 0,1% casminoacids. Another problem we faced is that RFP is also excited in the wavelength we use for GFP, making our measure a little bit inaccurate. A solution to this may be insert the bistable in the bacterial chromosome, using the Mini-Tn7 based system my team-mate has developed for biobricks. The less copies of the construction, the better the regulation is, because there is more repressors for each copy of the promoters.

Two repetitions of the switch were made, giving unequal results as it is shown in the graphs. We expect this great variation to be reduced in the improved models we are developing.

As is it shown in the charts, the switch engaged by temperature is quicker and stronger than the one elicited by IPTG. Temperature changes the structure of all the cI repressor instantly, while the IPTG takes some time to enter the cell and bind to the lacI repressor.

We’ve only tried the stability of only the RFP state, finding out it last 16 h at least. This effect is very noticeable when the growth takes place in a plaque, where you can see the change of colour without UV-light.