Team:DTU-Denmark/Switch
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UNDER CONSTRUCTION Biological switchesA 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. Bistable SwitchesNow, 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. Design of our Bi[o]stable SwitchThe 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 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). Step-wise Engineering of the SwitchIn our bistable switch we have three elements of regulation:
Step 1: The promoters and the repressorsThe 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.
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.
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-repressorsThe 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.
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-sitesTo avoid leakiness of the system we introduce a terminator downstream of the repressors, see figure 5.
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.
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 designThe 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.
Step 5: Inducing the systemNow 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.
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. ApplicationsIf 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.
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. |