UNDER CONSTRUCTION

What is a biological switch?

A biological switch is a system that enables cells to "remember" a state set by transient signals. This is important biologically because in cases such as differentiation of cells during development, gene regulatory systems must hold the state set during development. This can be accomplished by a network of genes that regulate one another through repressor and activator protein that they encode.

Design of our Bi[o]stable Switch

The simplest of such biological switches is one in which each of two repressor proteins represses the synthesis of the other. When both the repressor proteins are allowed to act, one of two stable states will be observed. In one steady state, the expression of repressor "one" is turned on and expression of repressor "two" is turned off. The repression of expression of repressor "two" is maintained by repressor "one", which means that the repressor "one" essentially acts as its own activator by inhibiting the expression of the repressor, repressor "two", that would repress its expression. In the other steady state, expression of repressor "two" is turned on and expression of repressor "one" is turned off.

In a system where the repressors can be controlled by outside input signals such as inducers or anti-repressor proteins, the system can be forced into its other stable state. This is illustrated in Figure 1.

We looked to nature for inspiration to design such a switch. The regulatory systems of the lambda phage as well as the Gifsy phages. The Gifsy phages are temperate phages found in Salmonella enterica that have an overall gene organisation typical of the lambdoid phage family (Regulatory Systems).

Step-wise Engineering of the Switch

The step-wise construction of our Bi[o]stable switch is demonstrated here, parts will be added to the switch as we build it up:

Step 1

The divergent promoters from both Gifsy1 and Gifsy2 phages are utilized in our system. The initial Gifsy1 and Gifsy2 constructs are illustrated below, Figure 2 and Figure 3, respectively.

Figure 2: The divergent promoters from the Gifsy 1 phage have been highlighted. When the Gifsy 1 phage repressor, GogR, is expressed, it will repress the pR1 promoter.

Figure 3: The divergent promoters from the Gifsy 2 phage have been highlighted. When the Gifsy 2 phage repressor, GtgR, is expressed, it will repress the pR2 promoter.

Figure 4: Both sets of divergent promoters have been highlighted. As illustrated, GogR (Gifsy 1 phage repressor) will repress the pR1 promoter when it is expressed. Transcription of gtgR is still allowed to some degree due to the pRM2 promoter. The strategy for the leakiness of pRM2 will be introduced later on.

Figure 5: As similarly demonstrated in Figure 4, both sets of divergent promoters have been highlighted. GtgR (Gifsy 2 phage repressor) will repress the pR2 promoter when it is expressed. Transcription of gogR is still allowed to some degree due to the pRM1 promoter. The strategy for the leakiness of pRM1 will be introduced later on.

Step 2

Figure 6: gifsy 1 and gifsy 2 are introduced in this image. gifsy 1 codes for the anti-repressor protein responsible for preventing GogR from binding to and thereby repressing the pR1 promoter. gifsy 2 codes for the anti-repressor protein responsible for preventing GtgR from binding to and thereby repressing the pR2 promoter.

Figure 7:

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Figure 11

These constructs are similar to the half switches seen in Figure 1, the difference being the presence of an extra promoter in front of each regulatory region. The "regulation" of this extra promoter will be described below. The Gifsy1 and Gifsy2 constructs are joined to form the basis of our bistable switch:

The step-wise construction of our Bi[o]stable switch is demonstrated here.

The Final Switch

Selection of Parts

Requirements before a biological switch functions. On the paper and theoretically.
Components of the switch
We have decided not to use cI, why? The hong kong paper flaws!! (REF) we did not use UV-activation why? To have a stable system we did not what to use cI and UV-regulatory systems as they can impose problems with the genetic stability.

Modeling

(modeling)

Selecting N protein and nut site

In the end, after evaluating what component pair to use we selected λ N-protein and nut-site. Different nut-sites N-protein systems have been identified and investigated (REFFF), the nutsites for λ-phage and p21, p22, are the best described (REFFF) comparison of the antiterminator effect have not been charfully investigated, as emphasis have been on function and interacting parts. we wanted to selected the nutsite with a strong consistent anti-terminator effect. But as this was not well defined continues work was done with the lambda nut side because more articles and knowledge was available, for potential trouble shooting and improvement of the system interaction and dynamic.

What have been described is that the N-nut-site pair have specific function and thus the λ-N-protein was used for continues, construction of the switch.

Fluorescent Proteins

as we departed in the idea from the terminator screening plasmids described in the partsregistry.org (REFFF) we had a primary focus on fluorescence proteins as our reporter systems. Further we wanted to have high quality data, with a high resolution. We descided on the in-house expertice on using a continous microfermentor system that can measure two fluorescence proteins continuously (biolector) and a flow cytometer, also capable of measuring at two different wave length.