CH391L/S14/Cellcountingmemory

Introduction
Memory in synthetic biology is the idea that a genetic circuit can have a sustained response to a temporary signal. Ajo-Franklin2007 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). Gardner2000 Since then, many different types of complex circuits involving memory have been developed. Inniss2013 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 naturally occurring memory circuits include the lambda phage switch, cellular differentiation, and cell division. Inniss2013

Mechanisms
There are many different methods of implementing memory in circuits. The majority are transcription-based, as the most-well understood naturally occurring memory circuit, the lambda phage switch, is transcription-based. Inniss2013 Within transcription methods, there are two main types: double-negative feedback loops and positive feedback loops. The double-negative feedback loops are based on two mutually repressing parts that are constitutively promoted. That way, either one can be on or the other, but not both at the same time. This creates a bistable circuit which will stay in whichever state is is put in, depending on the input. The simplest model for a positive feedback loop would be one input causing transcription of part A, which causes transcription of part B, which causes transcription of part A, and so on, keeping the circuit in an "on" state. A second input represses B, which causes the feedback loop to stop, keeping the circuit in an "off" state. Because the positive feedback loop will keep the "on" state on and the "off" state is just the resting state, neither will change if the input is applied, even if the input is removed. Another transcription-related mechanism includes using invertases, with each state being based on how the DNA is oriented. There are other possible methods of attaining memory in circuits using post-transcription or translation based mechanisms, but they are more difficult to design and implement. The idea of using post-transcriptional RNA modification to create memory has been proposed, such as a mechanism to make a positive feedback loop through polyadenylation of RNA (which makes the RNA more likely to be translated) that codes for a polyadenylation protein. Another possible method would be using kinases to create a phosphorylation-based system of controlling signal output. This is something that occurs naturally, though it is harder to design in synthetic circuits.

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. Gardner2000 Because the circuit will stay in one state even when the input is removed, the circuit has a form of memory. 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 used to give circuits memory. In order to make the circuit respond after a set number of signals, the circuit must first remember how many signals there have been, meaning that memory is intrinsic to counters. One application of counters would be causing cell death after a certain number of cell divisions. Smolke2009 For example, the cell could have a counting mechanism that responds to a molecule related to cell division, and when the counter reaches a certain point, the counter would cause cell death using a toxin or some other mechanism. This could be used as a regulatory method of keeping engineered bacteria from proliferating if they escape, though there are problems associated with it, namely the possibility of mutations in the counter or cell death function. Wright2013

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. Friedland2009 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. Figure 1 shows designs for a two-counter and three-counter. For both counters, the first coding region has a constitutive promoter, PLtet0-1, and all coding regions have a cis-acting repressor (cr) that forms a stem-loop structure that prevents translation. They then use a trans-acting noncoding RNA (taRNA) to bind with the cr and allow translation, which is promoted using arabinose (the input signal being counted). For a two-counter, the first coding region is for T7 RNA polymerase (RNAP), and the second is for green fluorescent protein (GFP) with a T7 promoter, meaning that the coding region will only be transcribed by T7 RNAP. Thus, the first signal pulse of arabinose allows for the translation of the T7 RNAP, and the second pulse allows the GFP to be transcribed, producing the output signal. The three-counter is similar, but the T7 RNAP translates T3 RNAP instead, which then translates GFP, meaning that three pulses of arabinose are needed to get the output. Friedland2009

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. Friedland2009 To make a three-counter, there would be three parts, each bookended with a different sequence that different invertases will recognize. For instance, the first part has two FRT sites facing, which the increase flpe will recognize, cutting that section of DNA out and putting it back in reversed. The first two parts code for invertases with terminators at the end that have the target sites they recognize inside them, which, when flipped, prevent further invertases from being produced, meaning that that section of DNA will stay flipped. In being flipped, the terminator is removed and an arabinose promoter that was on the opposite side is added to the front of the next part. The final part is the GFP coding region, which after the second part is flipped, will be promoted by arabinose. A similar setup uses the same invertases and GFP region, but the promoters use different molecules to activate transcription, meaning it needs to count three different molecules in a specific order to get the output.

Inversion Switch
A more complex circuit that has been attempted is another switch based on invertases that takes in two inputs and gives a different response depending on the order the inputs were added. Ham2008 By using two invertases and staggering the target sites so that the sequences that get flipped overlap, Ham et. al designed a circuit that would express two distinct outputs based on the order of inputs added. Figure 4 shows a hypothetical circuit using this design, where the addition of promoter B before promoter A causes a different end result from the addition of promoter A before promoter B. This is accomplished by placing terminators on both sides of the middle sequence so that the final product on each side will be blocked from transcribing until one of those two conditions has been met. Ham et. al used two specific invertases, hin and tim, as they are orthogonal, inducible, well studied, and the size of the DNA being flipped is flexible. When actually attempted, this did not work ideally. The ivertases equilibrated, meaning that after enough time passed from adding a given input, the two possible states (the starting state and the final state) were present in equal proportions. Because each output is only seen after adding each invertase once, the final output was only be seen in roughly a fourth of the colonies. Additionally, one of the outputs was not seen at all, most likely due to poor transition rates of the fim invertase for unknown reasons. Despite the problems, parts of the device are sound, and further development using these ideas is possible. Ham2008

iGEM
Because it is so ubiquitous in digital circuits, memory in genetic circuits is an obvious idea for iGEM teams, and numerous devices have been made involving memory. 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. Lou2010 The Brown University Team created a light-activated circuit based on the switch designed by the Peking team. Also, the Bologna iGEM team 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. Inniss2013 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. Inniss2013 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. Inniss2013