Team:DTU-Denmark/Switch

From 2010.igem.org

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<p align="justify">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 <i>Salmonella enterica </i> that have an overall gene organisation typical of the lambdoid phage family (for more theory please see <a href="https://2010.igem.org/Team:DTU-Denmark/Regulatory_sytems" target="_blank" >Regulatory Systems</a>).</p>
<p align="justify">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 <i>Salmonella enterica </i> that have an overall gene organisation typical of the lambdoid phage family (for more theory please see <a href="https://2010.igem.org/Team:DTU-Denmark/Regulatory_sytems" target="_blank" >Regulatory Systems</a>).</p>
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<p align="justify">The use of Gifsy phage key regulation elements in our switch has several advantages compared to lambda regulatory elements. One advantage is that in Gifsy it is possible to monitor both repressor and anti-repressor expression at the DNA level by inserting a reporter gene downstream of each gene. The assumption is that the measurable reporter gene concentration corresponds to the repressor and anti-repressor concentrations. This assumption does not take posttranslational protein modifications, degradation events or different half live times of the proteins into account, but it will give us an approximated concentration. With this approach it would be difficult in lambda to monitor repressor expression and concentration by a reporter gene after a SOS induced cleavage of the repressor itself. Here it would be necessary to measure the repressor concentration directly on protein level or the reporter gene has to be engineered in such a way, that it also will be cleaved after a SOS response, so both proteins are affected. Another possibility is to fuse the reporter gene directly to the gene of interest, but this gives rise to yet other problems e.g. protein is not biological active due to disrupted conformation or degradation behavior could be changed.</p>
 
<h1>Step-wise Engineering of the Switch</h1>
<h1>Step-wise Engineering of the Switch</h1>
<p align="justify">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:</p>
<p align="justify">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:</p>

Revision as of 09:12, 21 October 2010

Welcome to the DTU iGEM wiki!


UNDER CONSTRUCTION

What is a biological switch?

A biological genetic 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. The simplest of such switches can be accomplished by a network of genes that regulate one another through repressor and activator protein that they encode.

Natural Genetic Switches

Temperate bacteriophages are classic examples of natural genetic switches as they have alternative life-cycles. The bacteriophages can choose between the lysogenic cycle and the lytic cycle. It should be noted that the genetic switch illustrated in Figure 1 is not a bistable switch as once the lytic cycle is induced, the lysogenic state cannot be reattained.

Figure 1: The two distinct development pathways of a prophage life cycle [3].
In the lytic pathway the phage uses the bacterial molecular machinery to make many viral copies for infection of other cells before lysing the host bacterium. In contrast to the lytic pathway, the phage integrates its DNA into the bacterial genome in the lysogenic pathway. The lysogenic states is very stable, which means that the prophage can be replicated along with the bacterial genome for generations.

Despite the stability of the lysogenic state, the lytic state is readily induced when the bacteria are irradiated with ultraviolet light.


Bistable Switches

As previously mentioned, the aim of our project is to design a bistable switch. A bistable

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 2.

Figure 2: Simple bistable switch.


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 (for more theory please see 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 3 and Figure 4, respectively.

Figure 3: 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 4: 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 5: 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 fact that GtgR does not also repress the pRM2 promoter.
Note:The strategy for the leakiness of pRM2 will be introduced later in Steps 3 and 4.


Figure 6: 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 fact that GtgR does not also repress the pRM1 promoter.
Note:The strategy for the leakiness of pRM1 will be introduced later in Steps 3 and 4.


Step 2

Figure 6: 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 fact that GtgR does not also repress the pRM1 promoter.
Note:The strategy for the leakiness of pRM1 will be introduced later in Steps 3 and 4.


Figure 7: 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 8: The action of the anti-repressor protein, Gifsy 2 from the Gifsy 2 phage is illustrated. Gifsy 2 binds to repressor protein GtgR thereby preventing its repressor action. This means that GtgR is no longer able to bind to the pR2 promoter leaving the expressed GogR to bind to the pR1 promoter.
Note: The strategy for the leakiness of the pRM2 promoter will be introduced in Steps 3 and 4.


Figure 9: The action of the anti-repressor protein, Gifsy 1 from the Gifsy 1 phage is illustrated. Gifsy 1 binds to repressor protein GogR thereby preventing its repressor action. This means that GogR is no longer able to bind to the pR1 promoter leaving the expressed GtgR to bind to the pR2 promoter.
Note: The strategy for the leakiness of the pRM1 promoter will be introduced in Steps 3 and 4.


Step 3

Figure 10: The nut sites from p22 and lambda phages are introduced into the construct (for theory see Regulatory Systems). These nut sites will contribute to the robustness of the switch as described in Step 4.


Step 4

Figure 11: The anti-terminator proteins, Gp22 and GpN are introduced. In this image, Gp22 has been expressed and by binding to the p22 nut site, it enables RNA polymerase to bypass the terminator and continue transcription. This contributes to the robustness of the bistable switch as even though the pRM1 promoter allow transcription through them. Transcription through the terminator is prevented without the presence of the anti-terminator thereby not allowing spontaneous change of states.


Figure 12: In this image, GpN has been expressed and by binding to the lambda nut site, it enables RNA polymerase to bypass the terminator and continue transcription. Transcription through the terminator by GpN allows continuous expression of anti-terminator as well as anti-repressor, which will keep the cell in the induced state and thereby red. Even though transcription through pRM1 is permitted and GogR is expressed, it will be derepressed by the anti-repressor thus inactivating GogR. The state is maintained and the cell emits geen light.


In the switch design, each half switch contains a nut site followed by a terminator, as well as an antiterminator. The roles of these parts are to increase the stability of the current state of the switch. The pRM promoters are not very well repressed by the GogR/GtgR repressors and promotes transcription even in their presence. If transcription was allowed to continue to the antirepressor located on the inactive switch, the switch could change state spontaneously. The terminator ensures that this does not happen. The antiterminator of the active state is expressed, allowing continued transcription past the terminator.

The Final Switch

Figure 13: Our final construct is illustrated.


Applications of our Bi[o]stable switch