Team:UT-Tokyo/Sudoku construct

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= '''Sudoku''' =
= '''Sudoku''' =
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<html>
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[https://2010.igem.org/Team:UT-Tokyo/Sudoku_abstract Introduction]   
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<div>
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System    
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        <ul id="inpagemenu">
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[https://2010.igem.org/Team:UT-Tokyo/Sudoku_lab_note  Lab note]             
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                <li><a href="/Team:UT-Tokyo/Sudoku_abstract" id="abstract">Introduction</a></li>
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[https://2010.igem.org/Team:UT-Tokyo/Sudoku_result  Result]
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                <li><span>System</span></li>
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                <li><a href="/Team:UT-Tokyo/Sudoku_modeling" id="modeling">Modeling</a></li>
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== '''Construct''' ==
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                <li><a href="/Team:UT-Tokyo/Sudoku_experiments" id="experiment">Experiments</a></li>
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                <li><a href="/Team:UT-Tokyo/Sudoku_perspective" id="perspective">Perspective</a></li>
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            <li><a href="/Team:UT-Tokyo/Sudoku_reference" id="reference">Reference</a></li>
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            </ul>
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</div>
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<div id="clear"></div>
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</html>
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== '''System''' ==
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'''Overall construct'''
[[Image:Sudoku_const_Main_4.png|680px|Sudoku's main construct (We are making more simple version)]]
[[Image:Sudoku_const_Main_4.png|680px|Sudoku's main construct (We are making more simple version)]]
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Here we will explain how our bacteria solve Sudoku.
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Now we'll explain how to solve Sudoku with ''E.coli''.
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First, we begin by preparing ''E.coli'' corresponding to each cell number. We make these bacteria “decide” what number they should differentiate into.
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During this process, patial distribution (the position in a 4×4 grid) is temporarily lost. Instead, spatial information is represented by genetic identities assigned to each bacterium.
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In fact, the solving process is not performed on a 4×4 grid, but instead in a flask where we mix bacteria possessing all 16 types of genetic spatial identity.
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While in this mixture, the puzzle is solved as a result of inter-bacterial interaction.
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(We will explain this later in detail.)
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To explain this, we divide several steps:
 
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# 1. [[#4C3 leak switch]]
 
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# 2. [[#Signal virus]]
 
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## * transcription of MS2 phage & location sequence
 
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## * infection of signal virus
 
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## * selective translational suppression
 
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# 3. [[#Visualization of results]]
 
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[[Image:Sudoku_system_1.png|200px|thumb|How do the bacteria “decide” what number they become?]]
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So, how do the bacteria “decide” what number they become?
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Let’s take a look at the figure.
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The number in cell 1-1 has not yet decided.
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There are bacteria differentiated as 1 in the same column, 3 in the same row, and 2 in the same block, so the bacteria representing cell 1-1 should differentiate into number 4.
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In this way, bacteria which are not assigned a number from the beginning have to decide the number they differentiate into by judging the identity of other bacteria.
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The 4C3 leak-switch we suggest enables such a system.
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In order to solve Sudoku by E. coli, each microbe should possess grid and number information, with the ability to transmit the information mutually. At the same time, it is also necessary to control which E. coli to receive the information, according to the grid information (e.g. the information of grid 1-1 can be received by E. coli in grid 1-2, but cannot be received by E. coli in 4-4 grid). We propose a new system for the information transformation by RNA virus; “signal virus”. In this system, we propose using four types of RNA phage each with number information from 1 to 4(namely, four types of messenger RNA of site-specific recombination enzyme (SSRE)).
 
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The point is that the grid information is not spatial one. Instead, information transmission is replaced by mutual interaction between genomes. The practical solvation is not done in the plate with 4x4 cells. It is actually solved by E. coli in each tube correspond to each grid, and the answer is induced by genome interactions in the tube.
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Let us explain this switch in detail.
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This switch outputs the number it did not receive when it receives the other three numbers.  
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For example, when the switch receives the numbers 1, 2 and 3, it outputs the number 4, and when it receives the numbers 2, 3 and 4, it outputs 1.  
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The switch must work despite the numbers not being received in a set order, or no matter how many times a number is received.
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In other words, it is required that the switch outputs a signal only after receiving three numbers,  regardless of the order of reception.
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This is the “4C3 leak-switch”.  
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So what is it, in physical terms, what we have been calling “numbers”?
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The numbers 1 through 4 are represented by 4 different kinds of recombinases.
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Bacteria communicate with each other by transmitting RNA encoding these recombinases,
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which are packaged in virus vectors.
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Look at the figure again.
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This communication system must be one that only conveys information between bacteria possessing identities of the same row, column or block.
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In this figure, bacteria with the cell identity 1-1 need to differentiate to “4”. Therefore they should not be allowed to receive information from bacteria with the cell identity 4-4 prompting others not to differentiate to “4”.
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The signal virus system together with the antisense RNA system enables such restricted communication to take place. We will discuss these systems in detail below.
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E. coli whose number is already determined (let's call them "uni-output state") transmit their number information using "signal virus" to those whose number is still not determined ("multi-output state"). Once those E. coli in “multi-output state” received number information from appropriate grids, they have to recognize and manage the information, and finally, output their apt number. For example, if E. coli in "multi-output state" received number information in the order like 1->2->3, then, it'll become "uni-output state" and output 4. If E. coli in "multi-output state" received number information in the order like 3->2->4, then, it'll output 1 and transmit the information to grids around it. Here, you have to be aware that in this system, we need to admit randomness in the order of number information E. coli receive, and detect the variation, not the frequency of the number information. Which means that, E. coli in "multi-output state" have to output 4 whether it received number information in the order 1->2->3 or 3->1->2, and have to remain in "multi-output state" whether it recieved information 2, or 1->2, or 1->2->2->2->1 in series. That is to say, E. coli should turn into "uni-output state" only when it recieved 3 different types of the number in any order, and output the left number information. This is what our "4C3 leak switch" realizes.
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The overall systems therefore are the following:
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#1. [[#4C3 leak-switch|The 4C3 leak-switch]] designates what number bacteria differentiate into.
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#2. [[#Genotype Specific Signal Transmitting Virus|The signal virus]] transmits this information to other bacteria.
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#3. [[#Antisense RNA|The antisense RNA system]] restricts exchange of information to that between bacteria possessing identities of the same row, column or block.
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Now we examine each of these systems in detail.
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== '''4C3 leak-switch''' ==
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== 4C3 leak switch ==
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[[Image:Sudoku_exp_1_1.png|150px|thumb|4C3 leak switch_1]][[Image:Sudoku_exp_1_2.png|150px|thumb|4C3 leak switch_2]]
[[Image:Sudoku_exp_1_1.png|150px|thumb|4C3 leak switch_1]][[Image:Sudoku_exp_1_2.png|150px|thumb|4C3 leak switch_2]]
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4Number information from appropriate grids is transmitted to E. coli by signal virus, which will be described later. 4 types of the number information (namely 1, 2, 3, 4) correspond to 4 types of messenger RNA which code different SSRE. Those messenger RNA will be carried by RNA phage to the inside of E. coli and being translated, and expressed as SSRE which corresponds to each number.
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The 4C3 leak-switch is a switch which receives three number signals and outputs the number signal that it did not receive, regardless of the order of reception. What we call number signals are mRNA encoding homologous recombinases. When three types of mRNA encoding the recombinases enter a bacterium, the switch turns on and transcription of the recombinase that it did not receive begins. The switch is composed of a promoter, the cre protein coding region and four terminators in between. Each terminator is flanked on both sides by the recognition sequence of a homologous recombinase, and a terminator is excised following expression of the corresponding recombinase. Therefore when the ''E.coli'' do not receive a number signal, they possess four terminators; when they receive one number signal, they have three terminators; and so on.  
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The structure of the 4C3 leak switch will be shown in the figure-. There are 4 terminators between promoter and coding regions which express proteins work as switch.
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The 4C3 leak-switch, as the name implies, is a switch that makes use of transcriptional leakage. We assembled the sequence so that when there is more than one terminator, cre protein is not expressed but it is when there is only one. In addition, a recombinase coding sequence flanking the terminator is excised at the same time. Therefore, when the recombinase 1 coding sequence is excised, terminator 1 is also excised. As a result, after a cell receives the number signals 1, 2 and 3, only the recombinase 4 coding sequence remains. Each cre protein expressed in this way irreversibility excises the sequence between the lox sites independently of the other species of cre proteins. In the example above, mRNA coding recombinase 4 corresponding to the answer of this particular puzzle, is transcribed. In conclusion, the 4C3 leak-switch begins transcription of the recombinase that it did not receive begins after receiving three signals. The state in which the bacteria transcribes a recombinase we called differentiated.
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Each of four terminators is inserted into a different kind of site-specific recombination array. When SSRE is expressed, it will bind on the specific array and cut the site. In this way, terminators are being cut off.
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== '''Genotype Specific Signal Transmitting Virus''' ==
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[[Image:Sudoku_exp_2.png|200px|thumb|Transcription of MS2 phage & location sequence]]
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The point is that each type of SSRE corresponds to each terminator. Thus, if there is no number information transmitted, there are still four terminators left before the protein cording region. If only one type of number information transmitted, which means that one type of SSRE came into the cell, the terminator correspond to the enzyme is cut off and there are still three of them left. In this way, if there are two types of number information being transmitted, tow terminators left, and if three types of number information being transmitted, only one terminators left. The maximum types of information E. coli in "multi-output state" can receive is three.
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The mRNA coding the homologous recombinases have two additional sequences attached allowing transmission of information to other bacteria. First, they contain a loading sequence to the MS2 phage which enables the mRNA to be attached to the coat protein of MS2 phage. This MS2 phage is the carrier that transfers the number signal to other bacteria. These phage are only synthesized after recombination takes place, therefore preventing undifferentiated bacteria from emitting a number signal precociously.
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Secondly, they contain a location sequence which assigns a cell number to the bacterium. We will explain this in detail later.  In conclusion, <br/>
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4C3 leak-switch activates<br/>
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→ transcription of mRNA coding a homologous recombinase<br/>
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→ production of MS2 phage protein <br/>
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→ production of phage with the number signal and location sequence attached to it<br/>
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In this way, differentiated E.coli produce phage with a number signal and location sequence and conveys this information to other bacteria.
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When 3 types of information are being transmitted, there are only one terminator left, which is not strong enough to stop all translations by RNA polymerase. Some RNA polymerases can "leak" through terminator, and this is why our switch is called "4C3 leak switch".
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== '''Antisense RNA''' ==
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Our terminator is designed as a very sensitive one. When there are more than two terminators, only few proteins coded ahead the terminator can be expressed, which is so few that it can't function well. Though, when there is only one terminator, RNA polymerase can leak on certain extent, which is enough for expressed proteins to work. By using this terminator, we can realize a switch which turns on when any three types of number information being transmitted.
 
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As you can see from the figure, actually, each corresponding SSRE coding region is near terminator, so it will be cut off together. If SSRE1 came into the cell, it will cut off SSRE1 coding region with terminator. Overall, when number information was transmitted in order like 1->2->3, and the switch turned on, there are only SSRE4 coding region left.
 
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When signal switch turns on, the left SSRE coding region (which is SSRE4 in the late example, and this will be the answer of SUDOKU), grid location coding region (location sequence), and phage protein coding region (MS2 gene cluster) will be translated. These proteins will be assembled and become RNA phage which transmits number information to other E. coli.
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The final component of our system, the antisense RNA system, restricts which bacteria are able to receive which number signals. The phage infect all bacteria, so there needs to be a way for the infected bacteria to shunt away mRNA not targeted to them. This is possible because bacteria representing each cell number transcribes a unique set of antisense RNA. This RNA is antisense to the location sequences included in the mRNA transferred by the phage. Each bacterium has a set of antisense RNA corresponding to the location sequence of bacteria from which are made not to receive a signal from. These antisense RNA attach to the mRNA, preventing transcription, allowing blockage of irrelevant number signals. For instance, bacteria with the identity 1-1 do not want to receive number signals from 1-1, 2-3, 2-4, 3-2, 3-3, 3-4, 4-2, 4-3, 4-4. Therefore, bacteria with identity 1-1 constitutively transcribe RNA antisense to the location sequence of these nine cell numbers and as a result do not receive the number signals from these cells. Thus this system disables bacteria to receive number signals not targeted to them, although the phage infect all bacteria.
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<gallery widths="170">
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Image:Sudoku_exp_3.png|Infection of signal virus.
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Image:Sudoku_exp_4_1.png|Selective translational suppression.
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Image:Sudoku_const_Antisense_2.png|The construct of selective translational suppression.
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</gallery>
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== '''Movie''' ==
<html>
<html>
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<object width="640" height="385"><param name="movie" value="http://www.youtube.com/v/rw3xpMsmJ1w?fs=1&amp;hl=ja_JP"></param><param name="allowFullScreen" value="true"></param><param name="allowscriptaccess" value="always"></param><embed src="http://www.youtube.com/v/rw3xpMsmJ1w?fs=1&amp;hl=ja_JP" type="application/x-shockwave-flash" allowscriptaccess="always" allowfullscreen="true" width="640" height="385"></embed></object>
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<object width="640" height="385"><param name="movie" value="http://www.youtube.com/v/x3IhEUcCV-k?fs=1&amp;hl=ja_JP"></param><param name="allowFullScreen" value="true"></param><param name="allowscriptaccess" value="always"></param><embed src="http://www.youtube.com/v/x3IhEUcCV-k?fs=1&amp;hl=ja_JP" type="application/x-shockwave-flash" allowscriptaccess="always" allowfullscreen="true" width="640" height="385"></embed></object>
</html>
</html>
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== Signal virus ==
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== '''Visualization of results''' ==
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When 4C3 leak switch got three types of number information and turns on, RNA phage with the left number information will be produced from the E. coli. This is the signal virus which transmits the number information to appropriate grids.
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=== Transcription of MS2 phage & location sequence ===
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[[Image:Sudoku_exp_2.png|200px|thumb|Transcription of MS2 phage & location sequence]]
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Every E. coli have a plasmid with a set of gene which codes information of grid which is not in the same law, column, and subgrid. Messenger RNA translated from this gene will be an antisense RNA of messenger RNA transmitted by RNA virus, and it is able to interrupt gene expression by making the double helix of messenger RNA and prevent ribosome to carry on protein expression. Using this system, no matter in case that E. coli is infected by RNA viruses of all grids, it can choose which information to receive. That's because if E. coli don't want to receive information from certain grid, it can make antisense RNA and stop the expression of the enzyme in RNA phage. For example, E. coli in grid 1-1 will have antisense RNA correspond to messenger RNA from grid 2-3, 2-4, 3-2, 3-3, 3-4, 4-2, 4-3, 4-4, and shut out number information from those girds.
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Thus, enzymes correspond to number information of appropriate grids are expressed in E. coli and launch the 4C3 leak switch.
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=== Infection of signal virus ===
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[[Image:Sudoku_exp_3.png|200px|thumb|Infection of signal virus.]]
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Infection of signal virus.
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Infection of signal virus.
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Infection of signal virus.
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Infection of signal virus.
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Infection of signal virus.
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Infection of signal virus.
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Infection of signal virus.
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Infection of signal virus.
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Infection of signal virus.
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Infection of signal virus.
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=== Selective translational suppression ===
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[[Image:Sudoku_exp_4_1.png|150px|thumb|Selective translational suppression.]]
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[[Image:Sudoku_const_Antisense_2.png|150px|thumb|The construct of selective translational suppression.]]
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Selective translational suppression.
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Selective translational suppression.
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Selective translational suppression.
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Selective translational suppression.
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Selective translational suppression.
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Selective translational suppression.
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Selective translational suppression.
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Selective translational suppression.
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Selective translational suppression.
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Selective translational suppression.
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<html>
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<object width="640" height="385"><param name="movie" value="http://www.youtube.com/v/oer8IR4g5W0?fs=1&amp;hl=ja_JP"></param><param name="allowFullScreen" value="true"></param><param name="allowscriptaccess" value="always"></param><embed src="http://www.youtube.com/v/oer8IR4g5W0?fs=1&amp;hl=ja_JP" type="application/x-shockwave-flash" allowscriptaccess="always" allowfullscreen="true" width="640" height="385"></embed></object>
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</html>
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== Visualization of results ==
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[[Image:Sudoku_const_Result.png|200px|thumb|The construct to visualize results.]]
[[Image:Sudoku_const_Result.png|200px|thumb|The construct to visualize results.]]
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In order to be able to "see" that SUDOKU has been solved, we will prepare cluster of E. coli for detection. This detection E. coli will shut out all information from RNA virus except the virus produced by itself. By using antisense RNA, they will express SSRE which corresponds to the answer E. coli introduced.
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At this point, you may be wondering how the results are visualized. The differentiation status of bacteria depends on which recombinase they possess. We need to convert this information into a visible form. To do this, we created detection bacteria which will be plated on a 4×4 grid. When these bacteria come in contact with the signal viruses, they are able to receive number signals and fluoresce a different color depending on the signal they receive. Thus, these bacteria recognize the type of number signal (the type of homologous recombinase) and express the corresponding fluorescent protein. As shown, the in plasmids of the detection bacteria a fluorescent protein coding sequence is placed downstream of the terminator, enabling it to be excised simultaneously as the terminator is excised. Therefore, after three signals enter the bacterium and three rounds of excision take place, only one fluorescent protein will remain. The remaining protein is expressed and its color signals to us which number it has differentiated into. For example, if cell 1-1 outputs number 1, only recombinase 1 is transcribed in the detection bacteria, which excises the terminator between the recognition sequences of recombinase 1 and therefore expresses Green Fluorescent Protein.
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In this detection E. coli, SSRE will cut off double terminator introduced ahead of fluorescent protein coding region. Thus, parallel fluorescent protein will be expresses according to the number information. For example, if the answer of grid 1-1 is 1, SSRE1 will be expressed inside the detection E. coli of the grid, and gfp, which is parallel fluorescent protein for 1, will be expressed.
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Those detection E. coli will be placed into the detection plate, an imitating plate of the actual 4x4 plate for SUDOKU. The final step is to pour the medium in which E. coli finished solving SUDOKU to the plate. Each detection E. coli in the grid will begin to express fluorescent protein corresponding to the number and we can "see" the final answer!
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== '''Combine all systems''' ==
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Now that we have discussed each component independently, let us recapitulate the flow of the entire process.  
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In Sudoku, there are cells which are filled from the beginning and ones which are not. To create a puzzle like the one represented above, bacteria in cell 4-4 need to differentiate into state 4. Therefore we need to infect the undifferentiated bacteria in cell 4-4 with phage possessing number signals 1, 2 and 3 in advance. In this way, if we create undifferentiated bacteria corresponding to each cell and phage possessing the relevant number signals in advance, we can create various “problems” of Sudoku. We place 16 types of bacteria, corresponding to each of the 16 cells, some of them differentiated from the beginning, into a flask to cultivate. During cultivation, the process of transferring number signals from differentiated bacteria to undifferentiated bacteria is repeated and eventually the number all bacteria differentiate into  is decided. When this happens, the supernatant of the culture contains phage that possess information on the answer to the puzzle. We extract these phage and spread them on the plate housing detection bacteria. The detection bacteria express a fluorescent protein depending on the number signal they receive, allowing visualization of the answer to the problem.
</div>
</div>
{{UT-Tokyo_Foot}}
{{UT-Tokyo_Foot}}

Latest revision as of 03:55, 28 October 2010

UT-Tokyo

Sudoku

System

Overall construct

Sudoku's main construct (We are making more simple version)

Here we will explain how our bacteria solve Sudoku.

First, we begin by preparing E.coli corresponding to each cell number. We make these bacteria “decide” what number they should differentiate into. During this process, patial distribution (the position in a 4×4 grid) is temporarily lost. Instead, spatial information is represented by genetic identities assigned to each bacterium. In fact, the solving process is not performed on a 4×4 grid, but instead in a flask where we mix bacteria possessing all 16 types of genetic spatial identity. While in this mixture, the puzzle is solved as a result of inter-bacterial interaction. (We will explain this later in detail.)


How do the bacteria “decide” what number they become?

So, how do the bacteria “decide” what number they become? Let’s take a look at the figure. The number in cell 1-1 has not yet decided. There are bacteria differentiated as 1 in the same column, 3 in the same row, and 2 in the same block, so the bacteria representing cell 1-1 should differentiate into number 4. In this way, bacteria which are not assigned a number from the beginning have to decide the number they differentiate into by judging the identity of other bacteria. The 4C3 leak-switch we suggest enables such a system.


Let us explain this switch in detail. This switch outputs the number it did not receive when it receives the other three numbers. For example, when the switch receives the numbers 1, 2 and 3, it outputs the number 4, and when it receives the numbers 2, 3 and 4, it outputs 1. The switch must work despite the numbers not being received in a set order, or no matter how many times a number is received. In other words, it is required that the switch outputs a signal only after receiving three numbers, regardless of the order of reception. This is the “4C3 leak-switch”.

So what is it, in physical terms, what we have been calling “numbers”? The numbers 1 through 4 are represented by 4 different kinds of recombinases. Bacteria communicate with each other by transmitting RNA encoding these recombinases, which are packaged in virus vectors. Look at the figure again. This communication system must be one that only conveys information between bacteria possessing identities of the same row, column or block. In this figure, bacteria with the cell identity 1-1 need to differentiate to “4”. Therefore they should not be allowed to receive information from bacteria with the cell identity 4-4 prompting others not to differentiate to “4”. The signal virus system together with the antisense RNA system enables such restricted communication to take place. We will discuss these systems in detail below.

The overall systems therefore are the following:

  1. 1. The 4C3 leak-switch designates what number bacteria differentiate into.
  2. 2. The signal virus transmits this information to other bacteria.
  3. 3. The antisense RNA system restricts exchange of information to that between bacteria possessing identities of the same row, column or block.

Now we examine each of these systems in detail.

4C3 leak-switch

4C3 leak switch_1
4C3 leak switch_2

The 4C3 leak-switch is a switch which receives three number signals and outputs the number signal that it did not receive, regardless of the order of reception. What we call number signals are mRNA encoding homologous recombinases. When three types of mRNA encoding the recombinases enter a bacterium, the switch turns on and transcription of the recombinase that it did not receive begins. The switch is composed of a promoter, the cre protein coding region and four terminators in between. Each terminator is flanked on both sides by the recognition sequence of a homologous recombinase, and a terminator is excised following expression of the corresponding recombinase. Therefore when the E.coli do not receive a number signal, they possess four terminators; when they receive one number signal, they have three terminators; and so on.

The 4C3 leak-switch, as the name implies, is a switch that makes use of transcriptional leakage. We assembled the sequence so that when there is more than one terminator, cre protein is not expressed but it is when there is only one. In addition, a recombinase coding sequence flanking the terminator is excised at the same time. Therefore, when the recombinase 1 coding sequence is excised, terminator 1 is also excised. As a result, after a cell receives the number signals 1, 2 and 3, only the recombinase 4 coding sequence remains. Each cre protein expressed in this way irreversibility excises the sequence between the lox sites independently of the other species of cre proteins. In the example above, mRNA coding recombinase 4 corresponding to the answer of this particular puzzle, is transcribed. In conclusion, the 4C3 leak-switch begins transcription of the recombinase that it did not receive begins after receiving three signals. The state in which the bacteria transcribes a recombinase we called differentiated.

Genotype Specific Signal Transmitting Virus

Transcription of MS2 phage & location sequence

The mRNA coding the homologous recombinases have two additional sequences attached allowing transmission of information to other bacteria. First, they contain a loading sequence to the MS2 phage which enables the mRNA to be attached to the coat protein of MS2 phage. This MS2 phage is the carrier that transfers the number signal to other bacteria. These phage are only synthesized after recombination takes place, therefore preventing undifferentiated bacteria from emitting a number signal precociously. Secondly, they contain a location sequence which assigns a cell number to the bacterium. We will explain this in detail later. In conclusion,
4C3 leak-switch activates
→ transcription of mRNA coding a homologous recombinase
→ production of MS2 phage protein 
→ production of phage with the number signal and location sequence attached to it
In this way, differentiated E.coli produce phage with a number signal and location sequence and conveys this information to other bacteria.

Antisense RNA

The final component of our system, the antisense RNA system, restricts which bacteria are able to receive which number signals. The phage infect all bacteria, so there needs to be a way for the infected bacteria to shunt away mRNA not targeted to them. This is possible because bacteria representing each cell number transcribes a unique set of antisense RNA. This RNA is antisense to the location sequences included in the mRNA transferred by the phage. Each bacterium has a set of antisense RNA corresponding to the location sequence of bacteria from which are made not to receive a signal from. These antisense RNA attach to the mRNA, preventing transcription, allowing blockage of irrelevant number signals. For instance, bacteria with the identity 1-1 do not want to receive number signals from 1-1, 2-3, 2-4, 3-2, 3-3, 3-4, 4-2, 4-3, 4-4. Therefore, bacteria with identity 1-1 constitutively transcribe RNA antisense to the location sequence of these nine cell numbers and as a result do not receive the number signals from these cells. Thus this system disables bacteria to receive number signals not targeted to them, although the phage infect all bacteria.


Movie


Visualization of results

The construct to visualize results.

At this point, you may be wondering how the results are visualized. The differentiation status of bacteria depends on which recombinase they possess. We need to convert this information into a visible form. To do this, we created detection bacteria which will be plated on a 4×4 grid. When these bacteria come in contact with the signal viruses, they are able to receive number signals and fluoresce a different color depending on the signal they receive. Thus, these bacteria recognize the type of number signal (the type of homologous recombinase) and express the corresponding fluorescent protein. As shown, the in plasmids of the detection bacteria a fluorescent protein coding sequence is placed downstream of the terminator, enabling it to be excised simultaneously as the terminator is excised. Therefore, after three signals enter the bacterium and three rounds of excision take place, only one fluorescent protein will remain. The remaining protein is expressed and its color signals to us which number it has differentiated into. For example, if cell 1-1 outputs number 1, only recombinase 1 is transcribed in the detection bacteria, which excises the terminator between the recognition sequences of recombinase 1 and therefore expresses Green Fluorescent Protein.

Combine all systems

Now that we have discussed each component independently, let us recapitulate the flow of the entire process. In Sudoku, there are cells which are filled from the beginning and ones which are not. To create a puzzle like the one represented above, bacteria in cell 4-4 need to differentiate into state 4. Therefore we need to infect the undifferentiated bacteria in cell 4-4 with phage possessing number signals 1, 2 and 3 in advance. In this way, if we create undifferentiated bacteria corresponding to each cell and phage possessing the relevant number signals in advance, we can create various “problems” of Sudoku. We place 16 types of bacteria, corresponding to each of the 16 cells, some of them differentiated from the beginning, into a flask to cultivate. During cultivation, the process of transferring number signals from differentiated bacteria to undifferentiated bacteria is repeated and eventually the number all bacteria differentiate into is decided. When this happens, the supernatant of the culture contains phage that possess information on the answer to the puzzle. We extract these phage and spread them on the plate housing detection bacteria. The detection bacteria express a fluorescent protein depending on the number signal they receive, allowing visualization of the answer to the problem.