Team:DTU-Denmark/Switch

A biological genetic switch is a system that enables cells to "remember" a state set by transient signals. Many biological processes exist in mutually exclusive states. Cells or organisms exist in one of the distinct states depending on the level of stimulus input. A change in states is triggered in response to fluctuations of a stimulus above or below threshold level. This is frequently mediated by a regulatory circuit with positive and/or negative feedback mechanisms.

Temperate bacteriophages are classic examples of such natural genetic switches as they can choose between two distinct life cycles, namely lytic and lysogenic. However, once the lytic cycle is instigated, the lysogenic cycle cannot be reinstated. For more information please click here.

Now, taking an idea from the world of electronics, we define a bistable switch with a memory function and with two mutually exclusive steady states. Once stability in one state is reached; only stimulus above threshold level would switch the system towards reaching the other steady state. Bistable switches in one state remain on when stimulus concentration decrease below the level originally required to flip them on in the first place. Such behavior is known as hysteresis.

However, bistable switches are not naturally occurring mechanisms. As in the example with bacteriophages, once the lytic cycle is induced, the lysogenic state cannot be reattained. This is also an example of a robust switch in which once the system is set to be in one state, reduction and in some cases even removal of the stimulus input does not trigger switching to the other state. Robust switches have biological importance because in mechanisms 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 proteins that they encode.

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.

Figure 1: Simple bistable switch.

We looked to nature for inspiration to design such a switch and found 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).

In our bistable switch we have three elements of regulation:

• divergent promoters with repressors from the Gifsy phages
• anti-repressors from the Gifsy phages
• antiterminators from the lambda phage

Step 1: The promoters and the repressors

The divergent promoters from the Gifsy phages are made up of a very strong pR promoter, and a less strong pRM promoter which controls expression of a repressor that represses the pR promoter (personal communication: Sebastien Lemire), see figure 2.

Figure 2: The two divergent promoters pR1 and pRM and the repressor GogR have been highlighted.

Since the divergent promoters do not have equal promoter strength, we decided to include two sets of divergent promoters from Gifsy1 and Gifsy2 respectively, see figure 3.

Figure 3: The divergent promoters from the Gifsy 1 and Gifsy 2 phages have been highlighted. The divergent promoters from Gifsy 1 are pR1 and pRM1, whereas the Gifsy 2 divergent promoters are pR2 and pRM2

The Gifsy1 phage repressor GogR will repress the pR1 promoter when it is expressed and the Gifsy2 phage repressor GtgR will repress the pR2 promoter when it is expressed. The weaker pRM1 and pRM2 are constitutive promoters and there will therefore be a constant expression of GogR and GtgR. Expression of both GogR and GtgR will result in repression of both pR1 and pR2. We will address this problem in step 2.

Because pR1 and pR2 are strong promoters they will be used in the switch to amplify the expression of the downstream genes from pRM2 and pRM1 respectively. Consequently the pR1 promoter will control half switch two (in figure 3 illustrated as the lower part) and pR2 will control half switch one (in figure 3 illustrated as the upper part).

Step 2: Introducing anti-repressors

The pR promoters can be de-repressed by adding anti-repressors to the system as these will hinder the effect of the repressors. The two anti-repressors AntO and AntT, will anti-repress GogR and GtgR respectively and thereby de-repress the pR1 and pR2 promoters.

Figure 4: When the anti-repressor is expressed it will bind to the repressor and thus expression from the pR promoter will occur. Here illustrated by the Gifsy2 system.

As illustrated in figure 4 the expressed anti-repressor AntO will anti-repress GogR and thereby cause de-repression of the pR1 promoter. As a consequence expression of the genes in half switch two are amplified. GtgR will then repress pR2. At this point there is still a low expression of GogR from the pRM promoter. We will adress this problem in step 3.

Step 3: Introducing the anti-terminators and the corresponding nut-sites

To avoid leakiness of the system we introduce a terminator downstream of the repressors, see figure 5.

Figure 5: The location of the terminators is shown in correlation with the promoters and the repressor. No expression of the anti-repressor occurs when the terminator is functional.

In order to control the terminators, and thereby the expression of the anti-repressors, we introduce anti-terminators which are located downstream of the terminators. The anti-terminators are able to bind to the nut-site located upstream of the terminator. When the anti-terminator binds to the nut-site it enables transcription to proceed past the terminator.

The anti-terminator GpN as well as its corresponding nut-site are a part of the lambda phage regulatory system. The anti-terminator Gp24 as well as its corresponding nut-site are a part of the p22 phage regulatory system. For further details see the theory behind the regulatory system.

Figure 6: The anti-terminator from half switch one is expressed and thus the expression of anti-repressor AntT occurs. This results in the stability of half switch one.

The anti-terminators and the nut-sites contribute to the stability of the active state. If transcription was allowed to continue to the anti-repressor located on the inactive half switch, the switch could change state spontaneously. The terminator ensures that this does not happen. The anti-terminator of the active state is expressed, allowing continued transcription past the terminator.

Step 4: The final design

The illustration in figure 7 shows the final design of the switch when half switch one is stable. The GogR repressor represses the pR1 promoter and thus expression of the genes downstream of the pRM2 promoter are solely controlled by this weak promoter. The expressed GtgR will be anti-repressed by the AntT and the genes downstream of the nut-site will not be expressed due to the terminator.

The expression of the anti-terminator Gp24 makes transcription through the terminator possible and hence the reporter GFP is expressed.

Figure 7: The final design of the switch. Half switch one is in a stable state and the reporter GFP is expressed.

Figure 8: The final design of the switch. Half switch two is in a stable state and the reporter RFP is expressed.

Step 5: Inducing the system

Now that we are able to keep the switch in each state stable, we also need a means to be able to change the state of the switch at will. To do so we introduce two input plasmids illustrated in Figure9. The plasmids contain the anti-terminators and anti-repressors of each state respectively, in front of two different inducible promoters.

Figure 9: Input plasmid containing your favourite inducible promoter, the anti-terminator corresponding to one half switch and the anti- repressor corresponding to the other half switch.

When the plasmid corresponding to state 1 is induced, the switch is forced into state 1. This happens because the state’ s antiterminator and antirepressor are produced. The antirepressor inactivates state 2’ s repressor, resulting in increased transcription through state 1. At the same time, the antiterminator allows transcription to continue past state 1’ s terminator, where the genes for the same anti-repressor and anti-terminator are located. Thus, with a sufficient amount of induction, the switch will have switched states.

If successfully engineered, this new technological tool could advance methods used in many different fields of biological science, such as environmental engineering, food applications and medico technology. A few suggestions of such advances are listed below.

• In manufacturing, it could be applied as a cheap proofreading or control mechanism in production plants. In such a case, the product can be made to change colors when exposed to the right or wrong treatment or even an unwanted biological infection or compound.
• In manufacturing or environmental technology, it could be used for error or leakage tracking in process plants or out in the environment. The product can act as biosensor for compounds that are difficult to measure.
• In food control, a small piece of tape containing the modified organism could be placed on food packaging to ensure that the food product has not been exposed to undesired conditions such as high temperatures or hazardous compounds. This would allow for easier control of food conditions.
• In medical application, an organism containing our switch could be ingested and thereafter be tested to see if the organism experienced a certain drug, or condition on the way through the intestinal track.
• In molecular or medical science, it could be used to investigate chemotaxis and biofilm formation. This can be achieved by exposing a biological matrix to different light sources, there will be developed layers in the matrix and movements and development can be tracked, in relation to the two different exposures and the time between them.

Our original project idea was developed around the last example, where light (red light at 660nm and blue light at 470nm) would be used as input to induce switching between the two stable states. The initial ideas behind this was to use the bacteria to create artistic drawing or to track movement in biofilm.

Based on this idea, as well as research of biological switches and the regulatory mechanisms found in phages, we discovered untapped potential in designing a biological switch.