Team:UT-Tokyo/Counter/Project/Project

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The word "counter" may remind you of the machine with which you can count the number of objects, such as persons and vehicles. Some people familiar with electronic circuits may remind of the logic circuit. In each case, the system is regarded as memory device that remember the number of inputs, which is important for our lives.

In the natural world, cellular counters also memorize the number of events. For example, there are telomere length regulation[1][2], cell aggregation[3][4], etc. Telomere length of Saccharomyces cerevisiae is regulated by the number of the Rap1 protein, indicating the existence of counting system. The cell aggregation size of Dictyostelium is regulated by counting factor (CF). CF counts the number of aggregating cells and negatively regulates the cell adhesion. In these ways, cellular counters are widely utilized for the regulation of biological systems.

With synthetic biological approach, Ari et al constructed a cellular counter termed the riboregulated transcriptional cascade (RTC) counter[5]. The state transition occurs after an arabinose induction (Fig. 1). The system is regulated by riboregulators. The Biobrick part of this cellular counter has already existed, which was constructed by Tokyo-Nokogen 2009 and was named BBa_K225002, BBa_K225003 [6].

In order to expand the function of this counter, we added "reset system". The reset system enables the transition from any state to the initial state after a particular input. The property is expected to apply for a deterministic finite automaton, which is the system developed from information science. Within the finite number of states, the system makes the transition to another state in response to a particular input.

As the key of its resetting mechanism, our counter utilizes the regulation system based on sigma factor and anti-sigma factor. Sigma factor is a subunit of RNA polymerase and help it bind to the specfic sequence of the promoter. Anti-sigma factor blocks sigma factors from interacting with RNA polymerase. The utilization of this regulation system brings about benefit for attempt to make automaton.Firstly, the number of states can be increased easily because what we have to concern is the combination of sigma and anti-sigma factor. Secondly, the crosstalk between sigma and anti-sigma factors can be circumvented even if you raise the number of states. That is because we can choose such combinations between sigma and anti-sigma factors that have little crosstalk.

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Fig. 1 The concept of RTC counter. After first, second and third induction of arabinose, the state of cells moves from 0 to 1, 1 to 2 and 2 to 3, respectively.

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Sigma factors and anti-sigmas are used for constructing the sigma-memory. Sigma memory is a genetic device which have two states and can memorize the state. Sigma-memory is applied for constructing resettable counter.

sigma factor

Sigma factors are promoter recognition subunits of RNA polymerase. A sigma factor is associated with a part of promoters. A sigma factor recruits RNA polymerase to its corresponding promoter and initiates transcription. Sigma factors have great variety. Almost all sigma factors do not have one-to-one correspondence to a promoter, but some of them have one-to-one correspondence. Thus, if only sigma factors which have one-to-one correspondence are used, a transcription activating system in which a sigma factor activate the transcription only from corresponding promote can be made.

<img src = "" class = "figure-height" /> <legend>Fig. sigma factor A sigma factor recruit RNA polymerase to its corresponding promoter. Then transcription starts.</legend>

Anti-sigmas are a protein which is related to transcriptional control by sigma factors. An anti-sigma inhibits the binding between RNA polymerase and sigma factor. Consequently, anti-sigmas repress transcription from the promoters which sigma factors initiate. In the same way as sigma factor, anti-sigmas have great variety and some anti-sigmas prevent only a specific sigma factors from transcriptional control. Therefore a transcription control system (i.e. not only activating but also repressing) can be constructed by using specific sigma factors and anti-sigmas which have one-to-one correspondence. [1]

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                               <legend>Fig. anti-sigmas An anti-sigmas physically blocks the binding between corresponding sigma factors and RNA polymerase. Consequently, transcription from corresponding promoter is inhibited.</legend>

sigma-memory construction

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                               <legend>Fig. construction of sigma memory</legend>

This is the construction of our sigma-memory. This genetic circuit is composed of three parts. The gene of a sigma factor is placed at the downstream of the promoter that is induced by a substance A and at the downstream of Psigma, which is a promoter induced by the sigma factor. The latter one forms a positive feedback circuit. The gene of the corresponding anti-sigma is placed at the downstream of the promoter which is induced by a substance B.

At first, sigma factors and anti-sigmas do not exist. Input A induces the sigma factor expression and the concentration of the sigma factor. Though sigma factors are subjects to degradation [2], the state where sigma factors exist remains after the induction of A finished since the positive feedback circuit produces sigma factor when sigma factor exist. After the induction of input B, anti-sigma is expressed and transcriptions from the corresponding promoters, including positive feedback circuit, is inhibited. Both sigma factor and anti-sigma are subjects to degradation［2］, so all of them are decomposed after some time and sigma-memory returns to its original state (i.e. sigma factors and anti-sigmas do not exist.) This states remains since no substance that affect transcription exist.

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                               <legend>Fig. sigma-memory Sigma-memory is a genetic device which have two states and can memorize the state. Induction of A or B changes the state.</legend>

The existence/absence of sigma factor can be regard as 1/0 of memory, and these values of memory can switch by input A or input B. Using the promoter which is corresponding to the sigma factor, the information whether the value of memory is 1 or 0 can be observed as reporter expression. For example, consider the circuits on the right. The reporter is expressed when sigma-memory's value is 1 (i.e. sigma factor exists).

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                               <legend>Fig. construction of sigma memory with reporter The value of sigma-memory can be observed as reporter expression. </b>

<img src = "" class = "figure" style="width:400px;float:left;"/> <img src = "" class = "figure" style="width:400px;float:left;" /> <legend style="clear:both">Fig. mechanism of sigma memory</legend>

In addition, sigma factors, promoters, and anti-sigmas which have one-to-one correspondence enable us to make multi-sigma-memory genetic circuits. To make the explanation easier, consider the case in which E. coli has two sigma memories, sigmaA-memory and sigmaB-memory. The value of sigmaA-memory change from 0 to 1 if input A1 exists and change from 1 to 0 if inputs B1 exists. Also the value of sigmaB-memory change from 0 to 1 if input A2 exists and change from 1 to 0 if input B2 exists. For example, after the induction of A1, only the value of sigmaA-memory change from 0 to 1 since sigmaA promotes only transcription from PsigmaA (promoter that is corresponding to sigmaA). Since the transcription from PsigmaB is not activated, sigmaB is not expressed and the value of sigmaB does not change. The same is true of input A2. After the induction of B1, anti-sigmaA is expressed and the value of sigmaA-memory changes from 1 to 0. However, anti-sigmaA has no effect on the transcription of PsigmaB and the value of sigmaB does not change. The same is also true of the input B2. Hence it can be confidently said that E. coli has two sigma memories.

<img src = "" class = "figure" /> <legend>Fig. Multi sigma-memory and information processing A example of information processing by a sigma-memory. Only after induction of A1 and A2 in this order, the value of sigmaB-memory changes from 0 to 1.</legend>

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Resettable counter is a genetic device which count the number of the induction event of arabinose. In addition the count can be reset to 0 by IPTG induction. In this section, the application of sigma-factor for constructing resettable counter is explained.

resettable counter construction

<img src = "" class = "figure" /> <legend>Fig. construction of resettable 2-counter The construction of resettable 2-counter, which can count the number of induction event of arabinose. The count can be reset by IPTG induction. The maximum number that this counter can count is 2.</legend>

The construction of our resettable counter is explained here.

The construction of our resettable counter is explained here. The maximum number this counter can count is 2. Sigma factors used in this construction are Ecf20_992 (Sigma1, BBa_K1461004) and Ecf11_3726 (Sigma2, BBa_K1461005). The promoter correspond to sigma1 and sigma2 are Pecf20_992 (Psigma1, BBa_K1461001) and Pecf11_3726 (Psigma2, BBa_K1461002), respectively. The corresponding anti-sigmas are AS20_992 (anti-1, BBa_K1461006) and AS11_3726 (anti-2, BBa_K1461007) respectively. These two sigma factors were chosen because they most strongly activate transcription from the corresponding promoter within sigma factors which satisfy following conditions. (1)Promoter-sigma factor pair and anti-sigma-sigma factor pair have one-to-one correspondence. (2)The anti-sigmas strongly repress the transcription from their corresponding promoter. (3)They and their corresponding anti-sigmas have negligible impact on growth of E. coli. (4) The sequence of their corresponding promoter conform to the standard of BioBrick. [1]

Cis-repressor sequence is crR12 (BBa_K1461000) and trans-activating RNA is taR12 (BBa_K1461003). This pair is selected because the leakage is minimum. ［4］The reporter in this construction is GFP(BBa_E0040).

mechanism and extension

At first, both the values of sigma1-memory and sigma2-memory are 0. Only the crRNA coding sigma1, which is at downstream of constitutive promoter is translated. After the first induction of arabinose, taRNA at the downstream of PBAD is transcribed and the crRNA coding sigma1 is translated, and the value of sigma1-memory changes from 0 to 1. Since sigma1 exists, crRNA coding sigma2 at the downstream of Psigma1 is transcribed. After the second induction of arabinose, taRNA is transcribed and sigma11 is expressed, and the value of sigma2-memory changes from 0 to 1. At this time, GFP at the downstream of Psigma2 is expressed and the number of count being 2 is observed.

After induction of IPTG, anti-sigma20 and anti-sigma11 are expressed, and the value of sigma20-memory and sigma11-memory changes to 0 (if the value of sigma memories are already 0, they remains 0). Therefore the count is reset.

<img src="" class="figure" /> <legend>Fig. mechanism of resettable counter</legend>

Since there are many kinds of pairs of sigma factors and corresponding promoters which have one-to-one correspondence, n-counter can be made by the same way. Besides, the construction of 2-counter can be simplified.

<img src = "" class = "figure" /> <legend>Fig. construction of simplified resettable counter The construction of resettable counter can be simplified. However, the simplified counter cannot reset from their final count.

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This simplified counter is made by only one sigma factor. It works as the same way as original 2-counter until 1 count. After 1 count, crRNA coding GFP is transcribed at the downstream of Psigma. Then when the next arabinose induction occurs, GFP is translated. This expression can be considered a report of 2 count. This simplified counter can be also extended to n count. The simplified counter can count up to larger numbers compared to the original counter even when the same number of sigma factors are used, but cannot be reset from their final count. We did experiments on this simplified counter.

comparison with previous counter

Our sigma-Recounter is improved version of the previous counter constructed by Ari.［5］In the previous counter, T7 RNA polymerase and T3 RNA polymerase is used as the counterpart of sigma factor in our counter Using sigma factors has two merits. One is the ability to make it easy to extend 2-counter for n-counter. The number of RNA polymerase derived from virus is limited, but there are many kinds of sigma factors. Consequently it is easier to construct n-counter by using sigma factor. Another is the existence of inhibitors. Anti-sigmas are inhibitors of sigma factors which has one-to-one correspondence. Inhibitor is necessary to realize reset function.

Other difference is the positive feedback circuits. Previous counter has no feedback circuits. Since sigma factor is more subject to degradation than RNA polymerase, the positive feedback circuit is necessary to keep "memory" (i.e. for sigma factor to remain) in our counter.

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In a previous study[1], many sigma and anti-sigma that can regulate transcription without crosstalk have been reported. Thus, a counter that has many states (count number)can be constructed. Even though in this project the reset function is simply a transition from other states to state 0, more general system that is capable of changing one state to any other states is possible. Such a system is already beyond a counter because it can have an input corresponding to any arbitrary transition between states. This system can be applied to algorithmic system, for example, biocomputer etc. Furthermore, a more general circuit that responds to more general inputs is possible to be considered by integrating additional circuits.

For example, the change of balance between 2 substances itself can be considered as a input. Considering substances A and B, this additional circuit is possible:

pA-repressorB-reporterA-activatorX-pX-repressorA

pB-repressorA-reporterB-activatorY-pY-repressorB

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Here, substances A/B activate promoter A/B. This additional circuit essentially contains a toggle switch structure with delay negative feedback loops. For example, when substance A becomes dominant against substance B, the toggle switch amplifies the dominance of promoter A and the following negative feedback suppresses the dominance. Consequently, this additional circuit is expected to convert the change of the dominance to a pulse expression of a reporter protein. Therefore, this additional circuit can expand the range of input.