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Team:Michigan/Future
From 2012.igem.org
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<h2>Positive Feedback Loop</h2> | <h2>Positive Feedback Loop</h2> | ||
| - | + | Leaky expression of each recombinase when under an inducible promoter may cause a significant fraction of the cell population to have a switch flipped in the wrong direction if the recombinase leakage is above the concentration threshold necessary to flip the switch. If neither of the recombinases are being induced, the only way the switch can be flipped is through leaky expression. In other words, the state of the switch may degrade over time into a mixed state. In order to remedy this problem, we propose a positive feedback mechanism. In addition to the desired genes to be expressed on either side of the switch, one could add the recombinase that will flip the switch back to its current state in the event that undesirable basal level expression of the opposite recombinase flips the switch. This will create a threshold level of recombinase activity that will need to be overcome by the opposite recombinase in order to completely flip the switch. Extra consideration would have to be given to the transcription and translation rates involved in a circuit like this. The threshold level of recombinase activity must not be so high that the opposite recombinase is unable to overcome this activity and flip the switch. Too high of a threshold may be a burden on the cell as well. On the other hand, the threshold level must not be so low that the basal level expression of the opposite recombinase is able to flip the switch to the wrong state. | |
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| - | [[File:posfeedback. | + | [[File:posfeedback.GIF|350px|thumb|center|In this figure, the system begins with a switching promoter that is sensitive to HbiF recombinase. Although the system has leaky expression of HbiF controlled by an inducible promoter (blue oval), its effects on the promoter (yellow arrow) and the flanking invertible regions (light blue and green triangles) are overwhelmed by activities of higher concentration of FimE recombinase. The higher concentration is achieved by producing FimE from the two sources: FimE expression driven by inducible promoter (yellow oval) and by the switching promoter (yellow rectangle). When an inducer is provided, HbiF expression is significantly increased (light blue oval), which outcompetes FimE activity and flips the promoter and its flanking invertible region. The system now contains IRR and IRL, invertible regions sensitive to FimE (half-blue, half-green triangles) flanking a switched promoter, and higher concentration of HbiF than FimE.]] |
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Alternatively, antisense RNA (asRNA) can be used to generate a threshold level of leaky recombinase gene transcription such that a higher level of recombinase expression is required before significant switching can occur. Expression of the competing recombinase can be reduced if downstream of the switching promoter is a sequence that produces low levels of mRNA complementary to the RBS of that competing recombinase, effectively acting as an asRNA. The competing recombinase must transcribe at higher levels before it can overcome the effects of asRNA. When the competing recombinase mRNA level passes the threshold level posed by the asRNA, recombinase proteins will be produced such that a switch occurs, at which time the asRNA of the now competing recombinase will be expressed. | Alternatively, antisense RNA (asRNA) can be used to generate a threshold level of leaky recombinase gene transcription such that a higher level of recombinase expression is required before significant switching can occur. Expression of the competing recombinase can be reduced if downstream of the switching promoter is a sequence that produces low levels of mRNA complementary to the RBS of that competing recombinase, effectively acting as an asRNA. The competing recombinase must transcribe at higher levels before it can overcome the effects of asRNA. When the competing recombinase mRNA level passes the threshold level posed by the asRNA, recombinase proteins will be produced such that a switch occurs, at which time the asRNA of the now competing recombinase will be expressed. | ||
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<h2>Fine tuning promoter and RBS strengths</h2> | <h2>Fine tuning promoter and RBS strengths</h2> | ||
Combination of promoter and RBS driving recombinase expression could also be fine-tuned such that each recombinase produces sufficient flipping when induced with minimal leakiness when not induced. | Combination of promoter and RBS driving recombinase expression could also be fine-tuned such that each recombinase produces sufficient flipping when induced with minimal leakiness when not induced. | ||
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<h2>Degradation Tags</h2> | <h2>Degradation Tags</h2> | ||
| - | + | One could imagine wanting to flip the switch in rapid succession. If the recombinase from a previous flipping is still present in the cell, this may delay the amount of time it takes to flip the switch to the desired state. In order to increase the ‘responsiveness’ of the circuit, degradation tags can be attached to the recombinases that will tell the cell to degrade them faster. These will prevent unwanted recombinases from sitting around in the cell too long. One must consider how this will affect flipping of the switch though. If the turnover rate of the recombinases are too high, there may not be enough time for them to flip the switch. | |
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<h2>Adding LRP and IHF sites</h2> | <h2>Adding LRP and IHF sites</h2> | ||
| - | + | NA-bending proteins such as leucine-responsive regulatory proteins (Lrp) anting the switching sequence, bringing IRR and IRL sites closer together. This additional component may increase the rate at which the tyrosine recombinases form a Holliday junction and flip the switching sequend integration host factor (IHF) protein natively interact with <i>fimS</i> in fimbriae producing <i>E. coli</i>. These proteins could bind close to and assist in creating a loop encapsulace (Holden et al., 2007). | |
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<h2>Future Impacts</h2> | <h2>Future Impacts</h2> | ||
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The ability to accurately control the onset and termination of expression for proteins of interest enables further automation on cell/virus-based protein synthesis systems, opening opportunities for novel or better optimized high or low-throughput applications. I imagine a simultaneous induced switch flipping in a 96-well microplate, which is particularly beneficial for time sensitive assays and also serves to reduce the number of samples required for meaningful statistical analysis. | The ability to accurately control the onset and termination of expression for proteins of interest enables further automation on cell/virus-based protein synthesis systems, opening opportunities for novel or better optimized high or low-throughput applications. I imagine a simultaneous induced switch flipping in a 96-well microplate, which is particularly beneficial for time sensitive assays and also serves to reduce the number of samples required for meaningful statistical analysis. | ||
| - | <h2> | + | <h2>References</h2> |
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| + | Holden et al., Microbiology (2007) 153(12) | ||
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Latest revision as of 20:59, 14 October 2012
Future Directions
Making a better switch
Push-button Switch
It is possible to construct the switch such that brief exposure with an inducer produces a burst of recombinase expression, which in turn act upon the switching sequence and forming a sustained switch. An example of such a mechanism would utilize constitutively expressed repressors to suppress recombinase expression until the inducer temporarily inhibits repression.Positive Feedback Loop
Leaky expression of each recombinase when under an inducible promoter may cause a significant fraction of the cell population to have a switch flipped in the wrong direction if the recombinase leakage is above the concentration threshold necessary to flip the switch. If neither of the recombinases are being induced, the only way the switch can be flipped is through leaky expression. In other words, the state of the switch may degrade over time into a mixed state. In order to remedy this problem, we propose a positive feedback mechanism. In addition to the desired genes to be expressed on either side of the switch, one could add the recombinase that will flip the switch back to its current state in the event that undesirable basal level expression of the opposite recombinase flips the switch. This will create a threshold level of recombinase activity that will need to be overcome by the opposite recombinase in order to completely flip the switch. Extra consideration would have to be given to the transcription and translation rates involved in a circuit like this. The threshold level of recombinase activity must not be so high that the opposite recombinase is unable to overcome this activity and flip the switch. Too high of a threshold may be a burden on the cell as well. On the other hand, the threshold level must not be so low that the basal level expression of the opposite recombinase is able to flip the switch to the wrong state.File:Posfeedback.GIF
In this figure, the system begins with a switching promoter that is sensitive to HbiF recombinase. Although the system has leaky expression of HbiF controlled by an inducible promoter (blue oval), its effects on the promoter (yellow arrow) and the flanking invertible regions (light blue and green triangles) are overwhelmed by activities of higher concentration of FimE recombinase. The higher concentration is achieved by producing FimE from the two sources: FimE expression driven by inducible promoter (yellow oval) and by the switching promoter (yellow rectangle). When an inducer is provided, HbiF expression is significantly increased (light blue oval), which outcompetes FimE activity and flips the promoter and its flanking invertible region. The system now contains IRR and IRL, invertible regions sensitive to FimE (half-blue, half-green triangles) flanking a switched promoter, and higher concentration of HbiF than FimE.
