Difference between revisions of "CH391L/S14/Cellcountingmemory"

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==Genetic Toggle Switch==
 
==Genetic Toggle Switch==
The first synthetic biological circuit to include memory was a genetic toggle switch, a circuit with two distinct states that would switch between the two based on the input.[2] In essence, all that is required is two pairs of constitutive promoters and repressors, along with the reporter gene that signals whether the switch is on or off. When one promoter is on, it induces transcription of the repressor for the other promoter, and vice versa. One of the promoters will also induce transcription of the reporter gene, while the other will not. That way, when one promoter is not being repressed, the other will be, and the reporter gene will either be on or off. This type of circuit exhibits bistability, where it will be in one of two stable states, depending on the environmental conditions (inputs). If the circuit is between the two states, it will naturally fall to one or the other, but it will not stay in the middle.
+
[[Image:CH3912014_Alex_G_toggle_switch_design.png | thumb | right | 400 px |Toggle switch design. Repressor 1 inhibits transcription from Promoter 1 and is induced by Inducer 1. Repressor 2 inhibits transcription from Promoter 2 and is induced by Inducer 2.[2]]]The first synthetic biological circuit to include memory was a genetic toggle switch, a circuit with two distinct states that would switch between the two based on the input.[2] In essence, all that is required is two pairs of constitutive promoters and repressors, along with the reporter gene that signals whether the switch is on or off. When one promoter is on, it induces transcription of the repressor for the other promoter, and vice versa. One of the promoters will also induce transcription of the reporter gene, while the other will not. That way, when one promoter is not being repressed, the other will be, and the reporter gene will either be on or off. This type of circuit exhibits bistability, where it will be in one of two stable states, depending on the environmental conditions (inputs). If the circuit is between the two states, it will naturally fall to one or the other, but it will not stay in the middle.
  
 
==Cell Counting==
 
==Cell Counting==
 
One of the clearest examples of the use of memory in genetic circuits is counting. Counting with genetic circuits involves sending an output when a certain stimulus has been inputted a set number of times. Counters are very useful in digital circuits by giving the circuit a form of memory, in that it can respond to a stimulus long after it was originally input by only responding after the stimulus has been input multiple times. This has been done with many different biological circuits, using oligonucleotide hybridization, recombination, transcription, phosphorylation, and RNA editing.[3]
 
One of the clearest examples of the use of memory in genetic circuits is counting. Counting with genetic circuits involves sending an output when a certain stimulus has been inputted a set number of times. Counters are very useful in digital circuits by giving the circuit a form of memory, in that it can respond to a stimulus long after it was originally input by only responding after the stimulus has been input multiple times. This has been done with many different biological circuits, using oligonucleotide hybridization, recombination, transcription, phosphorylation, and RNA editing.[3]
  
==Transcriptional Counter==
+
===Transcriptional Counter===
 
To make a counter based on transcription, there are a few different methods that have been developed. One possible method involves using the same promoter to induce transcription of different stages in a circuit.[4] That way, a short burst of the input will cause the first part to be transcribed, but the next parts cannot be transcribed until the first has been. Then, another short burst will cause the next part to be transcribed, as the first part already has been, and so on. This can be modified to count different numbers, and was done with two- and three-counters. The final output will not be seen until every part has been transcribed, meaning that the correct number of signals has been inputted. This way, the input signals have been counted.
 
To make a counter based on transcription, there are a few different methods that have been developed. One possible method involves using the same promoter to induce transcription of different stages in a circuit.[4] That way, a short burst of the input will cause the first part to be transcribed, but the next parts cannot be transcribed until the first has been. Then, another short burst will cause the next part to be transcribed, as the first part already has been, and so on. This can be modified to count different numbers, and was done with two- and three-counters. The final output will not be seen until every part has been transcribed, meaning that the correct number of signals has been inputted. This way, the input signals have been counted.
  
==DNA Invertase Cascade Counter==
+
===DNA Invertase Cascade Counter===
 
Another counting method involves using DNA recombinases that will invert a section of DNA between two oppositely oriented cognate sites in such a way that a DNA sequence with inverted regions will not be fully read until a certain number of inputs have been taken in.[4] For instance, to make a three-counter, the DNA sequence would include a part that has the DNA recombinase with a terminator at the end and sites surrounding it such that it will flip itself at the first signal, breaking the transcription of the recombinase and adding a promoter for the signal to the second part. After that, a different recombinase (such that it will not flip the first part back to its original conformation) with the same set up will be read with the second signal without being stopped by the terminator from the first, and will again flip itself, preventing the recombinase from being transcribed and adding a promoter for the input signal to the final part. Finally, at the third signal pulse, the last part will be transcribed and the output signal can be seen. Thus, it measures when the input signal has been received three times. This method can also be made different lengths, with a different number of DNA recombinase parts that will invert themselves in order to count to different numbers.
 
Another counting method involves using DNA recombinases that will invert a section of DNA between two oppositely oriented cognate sites in such a way that a DNA sequence with inverted regions will not be fully read until a certain number of inputs have been taken in.[4] For instance, to make a three-counter, the DNA sequence would include a part that has the DNA recombinase with a terminator at the end and sites surrounding it such that it will flip itself at the first signal, breaking the transcription of the recombinase and adding a promoter for the signal to the second part. After that, a different recombinase (such that it will not flip the first part back to its original conformation) with the same set up will be read with the second signal without being stopped by the terminator from the first, and will again flip itself, preventing the recombinase from being transcribed and adding a promoter for the input signal to the final part. Finally, at the third signal pulse, the last part will be transcribed and the output signal can be seen. Thus, it measures when the input signal has been received three times. This method can also be made different lengths, with a different number of DNA recombinase parts that will invert themselves in order to count to different numbers.
  
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#inniss2013 Inniss, M.C., Silver, P.A. (2013) Building Synthetic Memory. ''Current Biology'' '''23''': 812-816.
 
#inniss2013 Inniss, M.C., Silver, P.A. (2013) Building Synthetic Memory. ''Current Biology'' '''23''': 812-816.
 
//Review article about memory in synthetic biology
 
//Review article about memory in synthetic biology
#Friedland2009 Friedland, A.E., Lu, T.K., Wang, x., Shi, D., Church, G., Collins, J.J. (2009) Synthetic gene networks that count. ''Science'' '''324''': 1199-1202.
+
#Friedland2009 Friedland, A.E., Lu, T.K., Wang, X., Shi, D., Church, G., Collins, J.J. (2009) Synthetic gene networks that count. ''Science'' '''324''': 1199-1202.
 
//Genetic circuits for counting
 
//Genetic circuits for counting
 
</biblio>
 
</biblio>

Revision as of 04:41, 24 March 2014

Contents

Introduction

Memory in synthetic biology is the idea that a genetic circuit can have a sustained response to a temporary signal.[1] Memory is a very useful feature of circuits that allows for larger and more complex systems. Attempts at creating biological systems that allow for the use of memory have been researched as early as 2000, with the creation of a genetic toggle switch (by having two distinct states that it will stay in, a transient signal will have a long-lasting effect).[2] Since then, many different types of complex circuits involving memory have been developed[3]. Building genetic circuits that use memory is very useful for exploring more complex types of circuits and underlying concepts of these circuits, as well as gaining a better understanding of natural biological mechanisms. Certain natural occurrences of memory circuits include the lambda phage switch, cellular differentiation, and cell division.[3]

Genetic Toggle Switch

Toggle switch design. Repressor 1 inhibits transcription from Promoter 1 and is induced by Inducer 1. Repressor 2 inhibits transcription from Promoter 2 and is induced by Inducer 2.[2]
The first synthetic biological circuit to include memory was a genetic toggle switch, a circuit with two distinct states that would switch between the two based on the input.[2] In essence, all that is required is two pairs of constitutive promoters and repressors, along with the reporter gene that signals whether the switch is on or off. When one promoter is on, it induces transcription of the repressor for the other promoter, and vice versa. One of the promoters will also induce transcription of the reporter gene, while the other will not. That way, when one promoter is not being repressed, the other will be, and the reporter gene will either be on or off. This type of circuit exhibits bistability, where it will be in one of two stable states, depending on the environmental conditions (inputs). If the circuit is between the two states, it will naturally fall to one or the other, but it will not stay in the middle.

Cell Counting

One of the clearest examples of the use of memory in genetic circuits is counting. Counting with genetic circuits involves sending an output when a certain stimulus has been inputted a set number of times. Counters are very useful in digital circuits by giving the circuit a form of memory, in that it can respond to a stimulus long after it was originally input by only responding after the stimulus has been input multiple times. This has been done with many different biological circuits, using oligonucleotide hybridization, recombination, transcription, phosphorylation, and RNA editing.[3]

Transcriptional Counter

To make a counter based on transcription, there are a few different methods that have been developed. One possible method involves using the same promoter to induce transcription of different stages in a circuit.[4] That way, a short burst of the input will cause the first part to be transcribed, but the next parts cannot be transcribed until the first has been. Then, another short burst will cause the next part to be transcribed, as the first part already has been, and so on. This can be modified to count different numbers, and was done with two- and three-counters. The final output will not be seen until every part has been transcribed, meaning that the correct number of signals has been inputted. This way, the input signals have been counted.

DNA Invertase Cascade Counter

Another counting method involves using DNA recombinases that will invert a section of DNA between two oppositely oriented cognate sites in such a way that a DNA sequence with inverted regions will not be fully read until a certain number of inputs have been taken in.[4] For instance, to make a three-counter, the DNA sequence would include a part that has the DNA recombinase with a terminator at the end and sites surrounding it such that it will flip itself at the first signal, breaking the transcription of the recombinase and adding a promoter for the signal to the second part. After that, a different recombinase (such that it will not flip the first part back to its original conformation) with the same set up will be read with the second signal without being stopped by the terminator from the first, and will again flip itself, preventing the recombinase from being transcribed and adding a promoter for the input signal to the final part. Finally, at the third signal pulse, the last part will be transcribed and the output signal can be seen. Thus, it measures when the input signal has been received three times. This method can also be made different lengths, with a different number of DNA recombinase parts that will invert themselves in order to count to different numbers.

iGEM

Because it is so ubiquitous in digital circuits, memory in genetic circuits is a common topic for iGEM teams. For example, the Peking University iGEM team created a push-on push-off genetic switch, similar to the genetic toggle switch made by Gardner et. al.[5] The Bologna iGEM team[6] attempted to create a flip-flop circuit, a bistable device that can serve as one bit of memory in digital circuits.

Future

While the uses of memory circuits in synthetic biology will continue to grow for more and more complicated circuits, the same technology could also have medical or industrial applications.[3] In the medical field, being able to have a protracted response to a single input could be very useful for drug delivery and diagnosis. They could even be used in such a way that they will respond to a stimulus from a certain illness with either a reporter or the necessary treatment to make treating diseases faster. Another possible use would be as an indicator of some disease-inducing agent, such as radiation, so that a doctor could see whether their patient had been exposed or not.[3] As for industrial uses, the ability to permanently induce cultures of bacteria to make the product of interest would make using bacteria much more efficient.[3]

References

  1. Ajo-Franklin, C.M., Drubin, D.A., Eskin, J.A. (2007) Rational design of memory in eukaryotic cells. Genes and Development 21: 2271-2276 [Ajo-Franklin2007]
    Technique for incorporating memory into circuits
  2. Gardner, T.S., Cantor, C.R., Collins, J.J. (2000) Construction of a genetic toggle switch in Escherichia coli. Nature 403: 339-342. [Gardner2000]
    First reported synthetic biology memory device
  3. Inniss, M.C., Silver, P.A. (2013) Building Synthetic Memory. Current Biology 23: 812-816. [inniss2013]
    Review article about memory in synthetic biology
  4. Friedland, A.E., Lu, T.K., Wang, X., Shi, D., Church, G., Collins, J.J. (2009) Synthetic gene networks that count. Science 324: 1199-1202. [Friedland2009]
    Genetic circuits for counting