Team:Michigan/Future

From 2012.igem.org

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<h2>Making a better switch</h2>
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<h1>Making a better switch</h1>
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<h4>Push-button Switch (merge with cheaper chem synthesis)</h4>
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<h2>Push-button Switch (merge with cheaper chem synthesis)</h2>
It is possible to construct the switch such that -A burst of inducer producing a burst of recombinase, causing a sustained switch,  instead of continuous expression of recombinase(s)
It is possible to construct the switch such that -A burst of inducer producing a burst of recombinase, causing a sustained switch,  instead of continuous expression of recombinase(s)
-
Positive Feedback Loop
+
<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. Therefore, given a long enough period of time without induction of either recombinase, a steady state of ‘ON’ and ‘OFF’ configurations will be reached that is dependent on the leakage rates and recombination rates of the recombinases. As an example, consider two inducible promoters with the same leakage rate and two recombinases with the same recombination rate. Under these conditions, 50% of the switches in the population will be in the ‘ON’ state and 50% will be in the ‘OFF’ state during steady state. 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. [Insert Figure] 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.  
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. Therefore, given a long enough period of time without induction of either recombinase, a steady state of ‘ON’ and ‘OFF’ configurations will be reached that is dependent on the leakage rates and recombination rates of the recombinases. As an example, consider two inducible promoters with the same leakage rate and two recombinases with the same recombination rate. Under these conditions, 50% of the switches in the population will be in the ‘ON’ state and 50% will be in the ‘OFF’ state during steady state. 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. [Insert Figure] 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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Antisense RNA (asRNA)
+
<h2>Antisense RNA (asRNA)</h2>
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Fine tuning promoter and RBS strengths
+
<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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Degradation Tags
+
<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.  
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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Recombination sites around the inducible promoters (not sure what to call this)
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<h2>Recombination sites around the inducible promoters (not sure what to call this)</h2>
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Adding LRP and IHF sites
+
<h2>Adding LRP and IHF sites</h2>
DNA-bending proteins such as leucine-responsive regulatory proteins (Lrp) and integration host factor (IHF) protein could bind close to and assist in creating a loop encapsulating 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 sequence.
DNA-bending proteins such as leucine-responsive regulatory proteins (Lrp) and integration host factor (IHF) protein could bind close to and assist in creating a loop encapsulating 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 sequence.
Holliday junction: (doi) Holden, N et al. Comparative analysis of FimB and FimE recombinase activity. 10.1099/mic.0.2007/010363-0
Holliday junction: (doi) Holden, N et al. Comparative analysis of FimB and FimE recombinase activity. 10.1099/mic.0.2007/010363-0
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Chromatin remodeling to control recombinases (Eukaryotes)
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<h2>Chromatin remodeling to control recombinases (Eukaryotes)</h2>
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Future impacts:
+
<h1>Future Impacts</h1>
Impacts on Medicine, cells/virus being able to make informed decisions/basic computations
Impacts on Medicine, cells/virus being able to make informed decisions/basic computations
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.
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<h1>Making More Complex Circuits</h1>
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Making more complex circuits
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N recombinase pairs gives 2^N states </p>
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N recombinase pairs gives 2^N states </p>
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Revision as of 19:09, 30 September 2012

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Future Directions

Making a better switch

Push-button Switch (merge with cheaper chem synthesis)

It is possible to construct the switch such that -A burst of inducer producing a burst of recombinase, causing a sustained switch, instead of continuous expression of recombinase(s)

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. Therefore, given a long enough period of time without induction of either recombinase, a steady state of ‘ON’ and ‘OFF’ configurations will be reached that is dependent on the leakage rates and recombination rates of the recombinases. As an example, consider two inducible promoters with the same leakage rate and two recombinases with the same recombination rate. Under these conditions, 50% of the switches in the population will be in the ‘ON’ state and 50% will be in the ‘OFF’ state during steady state. 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. [Insert Figure] 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.

Antisense RNA (asRNA)

Fine tuning promoter and RBS strengths

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.

Degradation Tags

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.

Recombination sites around the inducible promoters (not sure what to call this)

Adding LRP and IHF sites

DNA-bending proteins such as leucine-responsive regulatory proteins (Lrp) and integration host factor (IHF) protein could bind close to and assist in creating a loop encapsulating 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 sequence. Holliday junction: (doi) Holden, N et al. Comparative analysis of FimB and FimE recombinase activity. 10.1099/mic.0.2007/010363-0

Chromatin remodeling to control recombinases (Eukaryotes)

Future Impacts

Impacts on Medicine, cells/virus being able to make informed decisions/basic computations 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.

Making More Complex Circuits

N recombinase pairs gives 2^N states

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