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| | <!-- Projectここから --> | | <!-- Projectここから --> |
| | <div id = "Project-block" style = "display:none"> | | <div id = "Project-block" style = "display:none"> |
| - | <!-- Project background --> | + | <!-- Project system --> |
| | <div id = "Project-1"> | | <div id = "Project-1"> |
| - | <img src = "https://static.igem.org/mediawiki/2014/d/db/Sub_background.png" class = "contTitle"/> | + | <img src = "https://static.igem.org/mediawiki/2014/8/8f/SubCTC_system.png" class = "contTitle"/> |
| - | <p>The word, "counter", will remind you the machine with which you can count the number of the objects, such as people and cars. Some people familiar with electronic circuits may remind the logic circuit. In any case, both are the memory device of the order of inputs and are important for our lives.<br /><br />Cellular counters also memorize the number of events. In nature, there are tellomere length regulation, cell aggregation and so on. <br />Ari et al constructed the cellular counter with synthetic biological approach, termed the riboregulated transcriptional cascade (RTC) counter[1].The state transition occurs after 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_K25003 [2]. <br />In order to expand the function of this counter, we added reset system. The reset sysytem realizes the movement from any state to the initial state after the particular input and is expected to movement from down stream states to upstream states by respective inputs, which we regard as automaton. Our counter utilizes the regulation system of sigma factor and anti-σ factor as the key of its resetting mechanism. In addition to reset function, the number of states can be increased more easily, because what we have to think is only the combination of σ and anti-σ factor, that is, we do not have to find new polymerase. Moreover, because there is orthonal relation between sigma and anti-sigma factor, we can choose combinations which help us avoid crosstalk problem. <br /><br />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.<br /><br />[1]Ari E. Friedland, et al., Science 324, 1199 (2009). <br />[2]https://2009.igem.org/Team:Tokyo-Nokogen/Parts</p> | + | <h3>EGP-2 promoter</h3> |
| - | </div>
| + | <p>EGP-2 promoter is the promoter of Epithelial cell adhesion molecule (EpCAM) protein.</p> |
| - | <!-- Project background --> | + | <p>EpCAM is a trans membrane glycoprotein which is expressed in epithelial cells and shows high level expression in various type of human epithelial carcinomas[2].Using this promoter, reporter genes can be expressed only in cancer cells.</p> |
| - | <!-- Project σ-memory -->
| + | <h3>miRNA-142</h3> |
| | + | <p>MiRNA is a class of non-coding RNA.MiRNA binds the miRNA-recognition element in the 3' untranslated region of the target gene. MiRNA shows the tissue-specific expression pattern. In our project, we use miRNA-142, which is expressed only in hematopoietic cells[3].Adding the recognition element of miRNA-142 in the 3' untranslated region of the reporter genes, leaky expression in non-carcinoma hematopoietic cells can be reduced.</p> </div> |
| | + | <!-- Project application --> |
| | <div id = "Project-2"> | | <div id = "Project-2"> |
| - | <img src = "https://static.igem.org/mediawiki/2014/e/e4/Sub_memory.png" class = "contTitle" /> | + | <img src = "https://static.igem.org/mediawiki/2014/b/b4/SubCTC_application.png" class = "contTitle" /> |
| - | <h3>σ factor</h3> | + | <p>We focused on a tissue-specific promoter and miRNA in order to detect CTCs.</p> |
| - | <p>σ factor is a subunit of RNA polymerase related to promoter recognitions. There are two types of sigma factors, one that is housekeeping (expressed constantly) and another that is expressed under some conditions. It is possible to deliver genes of sigma factors that are not derived from some species to another species, so we can use many kinds of σ factor. Which promoter RNA polymerase tends to bind to is decided by which σ factor RNA polymerase is bound. Especially some σ factors promote only transcription of a specific promoters. Therefore if we choose a set of sigma factors and promoters skillfully, we can control transcription without crosstalk. Here, “without crosstalk” means every σ factor in the set promotes only transcription of the cognate promoter.<br />Anti-σ is also a protein which is related to transcriptional control by σ factors. Anti-σ prevents σ factors from binding to RNA polymerase. Similarly as σ factor, there are many kinds of anti-σ and we can choose a set of σ factors, promoters, and anti-σs which has no crosstalk. In our project, we use such a set of σ factors, promoters, and anti-σs.</p> | + | <p>As well as hematopoietic cells, many other tissues shows specific miRNA expression patterns[3]. Therefore, if by making a circuit that returns an output to a certain miRNA expression pattern, we may identify where a CTC comes from.</p><br /> |
| - | <h3>σ-memory construction</h3>
| + | <p>In addition to the detection of CTCs, combinations of tissue-specific promoter and miRNA can be applied to many treatments. For example, suicide genes can be expressed in cancer cells in a specific tissue.</p> |
| - | <p>This is the construction of our σ-memory. If input A exists, σ factor is expressed. Then the positive feedback circuits of σ factor starts producing σ factor, and consequently σ factor will remain. Though if input B exists, anti-σ is expressed and the positive feedback circuits is inhibited. Both σ factor and anti-σ are subjects to degradation, so all of them are decomposed after some time and σ-memory returns to original state.<br />We can regard existence/absence of σ factor as 1/0 of memory, and this values of memory can switch by input A or input B. Using the promoter which is cognate to the σ factor, the information whether the value of memory is 1 or 0 can be derived. For example, consider the circuits on the right. The repotor is expressed if and only if σ-memory's value is 1 (i.e. σ factor exists).<br />In addition, no crosstalk sets of σ factors, promoters, and anti-σs enable us to make multi-σ-memory gene circuits. To make the explanation easier, consider the case in which E. coli has two σ memories, σA-memory and σB-memory. The value of σA-memory change from 0 to 1 if input A1 exists and change from 1 to 0 if inputs B1 exists. Also the value of σB-memory change from 0 to 1 if input A2 exists and change from 1 to 0 if input B2 exists. If the four input A1, B1, A2, and B2 have no crosstalk, σA-memory and σB-memory also have no crosstalk. For example, when input A1 exists, only the value of σA-memory change from 0 to 1 since σA promotes only transcription from PσA (promoter that is cognate to σA), and has no effect on σB. The same is true of input B1 and anti-σA (anti-σ that is cognate to σA). So it can be confidently said that E. coli has two σ memories.<br />Since this construction has a positive feedback, leakage of promoter may be a serious problem. If the promoter has leakage and σ factor is expressed when input A dose not exist, this error is enlarged by positive feedback. However if the leakage of anti-σ is satisfactory large, σ factor dose not produced from positive feedback when leak of σ factor occurs. Therefore leakage is not an obstacle of our project if we choose the promoter skillfully. | + | |
| - | <h3>σ-memory and automaton</h3>
| + | |
| - | <p>Speaking easily, an automaton is referred to what has states and transition rules. Every state has a transition rule, say it is decided what is the next state if an input exists. For example, the automaton on the right has five states, 0, 1, 2, 3, 4, and transition rules are represented by arrows. If the current state is 0 and the input is b, the next state is 2. Transition rules having loops or feedbacks are allowed.<br />σ-memory can be considered an automaton. Every tuple of σ-memories' value (say, (1, 1, 0, 1, 0)) corresponds to a state. It is possible to use one σ-memory as an inputs of another σ-memory. For example, consider the gene circuit on the right.The value of σA-memory( a σ-memory which uses σA in the construction as a σ factor) change from 0 to 1 if arabinose exists, also the value of σB-memory change from 0 to 1 if the value of σA-memory is 1 and IPTG exists. (This condition can be written in terms of mathematical logic, namely AND(σA, IPTG)) These gene circuits can be considered as the automaton on the right. Gene circuits which work as logic circuits (says AND, OR, NAND, etc...) have been designed.</p>
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| | </div> | | </div> |
| - | <!-- Project σ-memory -->
| + | </div> |
| - | <!-- Project Application -->
| + | <!-- Result --> |
| - | | + | <!-- Lab Data --> |
| - | <img src = "https://static.igem.org/mediawiki/2014/9/93/Sub_appli.png" class = "contTitle" /> | + | <div id = " Lab- block" style = "display:none;"> |
| - | | + | <div id = "Lab-1"> |
| - | <h3>resettable counter by σ-memory</h3> | + | </div> |
| - | <p>Since a counter is one kind of automata, σ-memory can be applied for making resettable counters. It can count the number of induction events of arabinose, and express a reporter correspond to each states. In addition, the count can be reset by IPTG induction.<br />2-counter is a counter that can count up to 2, and is an automaton that has three states, 0, 1, and 2. This automaton can be constructed by σ-memory as following. Two kinds of σ factor are necessary for constructing the 2-counter, so for the convenience we will call these two kinds of σ factors σA and σB. In state 0, both σA and σB do not exist (i.e. σA-memory and σB-memory is 0). In state 1, only σA exists and in states 2, both σ factors exist. The input is the induction of arabinose or IPTG, and the transition rules with it are showed in the Figure.<br />A gene circuit that realizes this automaton can be represented as following. The value of σA changes from 0 to 1 after arabinose induction (i.e. σA is expressed when arabinose exists). This change corresponds to the transition from state from 0 to 1.The value of σB-memory changes from 0 to 1 when both arabinose and σA exist. 【説明不足】Therefore the AND function is necessary. This change corresponds to the transition from state 1 to state 2. Both the value of σA-memory and σB-memory change from 1 to 0 after IPTG induction (i.e. anti-σA and σB is expressed when IPTG exist).</p>
| + | </div> |
| - | | + | <!-- Modeling --> |
| - | <h3>riboregulator</h3>
| + | <div id = "Modeling-block" style = "display:none;"> |
| - | <p>According to the discussion in 2-1, it is necessary to use AND function for the construction of a resettable counter. Therefore a riboregulator is used for the realization of the AND function.<br />A riboregulator is a post-transcriptional regulation system composed of two kinds of RNAs. Cis- repressed mRNA(crRNA) forms a stem-loop and its ribosomal binding site (RBS) is covered. Consequently the gene coded in cis-repressed mRNA isn't translated. However if trans-activating RNA (taRNA) exist, crRNA and taRNA are hybridized and RBS gets exposed and translation starts.<br />Namely, the gene coded in crRNA is expressed only when both crRNA and taRNA is transcripted. Therefore this system can be considered as the AND function. For example, consider the gene circuit, Plac-taRNA-d.term-Pbad-cr-RBS-GFP-d.term.(cr is the sequence in crRNA which binds to RBS) If and only if both IPTG and arabinose exists, GFP is expressed. Therefore, the riboregulator can combine two promoters and produce AND functions</p> | + | <div id = "Modeling-1"> |
| - | <h3>resettable counter construction</h3> | + | </div> |
| - | <p>The construction of our resettable counter is explained here.<br /><br />σ20 and σ11 are used as σA and σB as mentioned above respectively. The promoters correspond to σ20 and σ11 are Pσ20 and Pσ11, and the anti-σs are anti-20 and anti-11 respectively. GFP is used as a reporter. <br />At first, both the value of σ20-memory and σ11-memory are 0. Only the crRNA coding σ20, 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 σ20 is translated, and the value of σ20-memory changes from 0 to 1. Since σ20 exists, crRNA coding σ11 at the downstream of Pσ20 is transcribed. After the second induction of arabinose, taRNA is transcribed and σ11 is expressed, and the value of σ11-memory changes from 0 to 1. At this time, GFP at the downstream of Pσ11 is expressed and we can check the count as 2.<br />After an induction of IPTG, anti-σ20 and σ11 are expressed, and the value of σ20-memory and σ11-memory will be 0. Therefore the count is reset.<br /><br />Since there are many kinds of pair of σ factors and cognate promoters which have no crosstalk, n-counter can be made by the same way. Besides, the construction of 2-counter can be simplified.<br />This simplified counter is made by only one σ factor. It works as the same way as original 2-counter unill 1 count. After 1 count, crRNA coding GFP is transcribed at the downstream of Pσ. Therefore 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. Simplified counter can count up to larger numbers compared to the original counter even when the same number of σ factors are used, but cannot be reset from their final count. We did experiments on this simplified counter.</p> | + | </div> |
| | + | <!-- Achievement --> |
| | + | <div id = "Achieve-block" style = "display:none;"> |
| | + | <div id = "Achieve-1"> |
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| | + | </div> |
| | + | <!-- Safety --> |
| | + | <div id = "Safety-block" style = "display:none;"> |
| | + | <div id = "Safety-1"> |
| | + | </div> |
| | + | </div> |
| | + | <!-- Attribution --> |
| | + | <div id = "Attribution-block" style = "display:none;"> |
| | + | <div id = "Attribution-1"> |
| | + | </div> |
| | + | </div> |
| | + | <!-- Human Practice --> |
| | + | <div id = "Humanpractice-block" style = "display:none;"> |
| | + | <div id = "Humanpractice-1"> |
| | </div> | | </div> |
| - | <!-- Project application -->
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| | </div> | | </div> |
| | <!-- Teamここから --> | | <!-- Teamここから --> |
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