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Basic Flip Flop. Overview

The basic electronic part in a computer is the flip-flop, a circuit that can have only two possible states: 0 and 1. These states don’t change unless a new input comes, making this device a good inspiration for a new expression system. In this system the addition of a chemical substance or a change in the environment would result in a response that would be maintained even if the stimulus is retired. But, how is this synthetic regulation pathway made? The structure is composed by two repressors and their correspondent promoters. Each repressor inhibits the expression of the other one and the expression of the repressor is coupled with different fluorescent proteins. In this case we used the promoter PL and its repressor cI with the Plac and lacI system. As reporters for each of the states, we have GFP and RFP. An example in nature is the regulation of lambda phague, when it infects a bacterium and has to decide between lytic or lysogenic cycle.

The repressor cI is a mutant version that doesn’t work at 42ºC. Then, the system responds to two different signals: change of temperature (expression of RFP) and addition of IPTG (expression of GFP). For this device to work properly we need a strain deficient in LacI, in our experiments we use MC4100.

These devices has been done and tested before (Gardner, 2000) but the objective of our project is to find a way to improve its performance, either through mathematical modelling or an experimental approach. The basic flip-flop has some flaws in its behaviour such as the long time induction by IPTG and the duration of the change of state. Describing how the bistable works would help the other subteams to see if their genetics constructions actually work as they expected.

What would be the applications of this device?

• It could be used as an expression system for genetic therapy. A usual vector would need the addition of the inducer for the whole life of the patient, which may cause health problems. This new approach allows that only one dose promotes the expression of the desired gene for a long time, leading to a reduction in the amount of inducer injected.

• Also biosensors seem like a plausible application, changing the system so it responds to different inducers. Toxics substances or pollutants may be good targets for the development of new regulatory pathways.