Team:NCTU Formosa/Project/Design





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Project</a> <div class="select_sub"> Abstract</a></li> Introduction</a></li> Design</a></li> Parts</a></li> New Idea</a></li> Safety</a></li> </ul> </li> </ul>

<a>Results</a> <div class="select_sub"> <li><a href="http://2009.igem.org/Team:NCTU_Formosa/WetLab/Labworks" target="_self">Lab works</a></li> <li><a href="http://2009.igem.org/Team:NCTU_Formosa/WetLab/Timer" target="_self">Timer</a></li> <li><a href="http://2009.igem.org/Team:NCTU_Formosa/WetLab/Counter" target="_self">Counter</a></li> <li><a href="http://2009.igem.org/Team:NCTU_Formosa/WetLab/Memory" target="_self">Memory</a></li> <li><a href="http://2009.igem.org/Team:NCTU_Formosa/WetLab/Results_new" target="_self">New Idea</a></li> </ul> </li> </ul> <li><a href="http://2009.igem.org/Team:NCTU_Formosa/Modeling" target="_self">Modeling</a></li> </ul> <li><a>Notebook</a> <div class="select_sub"> <li><a href="http://2009.igem.org/Team:NCTU_Formosa/Notebook/Calendar" target="_self">Calendar</a></li> </ul> </li> </ul> <li><a href="http://2009.igem.org/Team:NCTU_Formosa/SubmittedParts" target="_self">Submitted Parts</a> </li> </ul> <li><a href="http://2009.igem.org/Team:NCTU_Formosa/Contact" target="_self">Contact</a> </li> </ul>

Design The main target in our project is to insert the sequences into the E.coli which serve the functions of both timer and bacteria counter. Once we turn on the device, the medium will become green. Afterwards, when the time is up or the concentration of bacteria invaded rises to a certain level, the medium will turn from green to red. The whole reaction can be divided into three stages: <ol> <li>Standby: The E.coli only grow but other specific metabolism is excluded.</li> <li>Lactose-accession: The lactose is added and the timer and the counter functions started.</li> <li>Lactose-consumption: When the lactose consumes or that the amount of the bacteria invaded is large enough, the alarm                                        system starts and the E.coli suicide later.</li> </ol> <object classid="clsid:D27CDB6E-AE6D-11cf-96B8-444553540000" codebase="http://download.macromedia.com/pub/shockwave/cabs/flash/swflash.cab#version=9,0,28,0" width="600" height="430" title="2009iGEM-principle_v3"> <param name="movie" value="http://2009.igem.org/wiki/images/1/17/2009iGEM-principle_v3.swf"> <param name="quality" value="high"> <embed src="http://2009.igem.org/wiki/images/1/17/2009iGEM-principle_v3.swf" quality="high" pluginspage="http://www.adobe.com/shockwave/download/download.cgi?P1_Prod_Version=ShockwaveFlash" type="application/x-shockwave-flash" width="600" height="430"> (1)	The Switch and The Input The lacI operon serves as the switch of our project, and lactose works as the starting signal. At the beginning, the constitutive promoter (<a href="http://partsregistry.org/Part:BBa_J23106" style="text-decoration:none;">BBa_J23106</a>) unceasingly produces the LacI protein (BBa_C0012), which represses the LacI promoter (<a href="http://partsregistry.org/Part:BBa_C0012" style="text-decoration:none;">BBa_R0010</a>). When fixed amount of lactose is added, the lactose-lacI complex releases from the promoter and causes the transcription and the translation of downstream sequences begin. (2)	The Timer Within the reaction, the lactose degrades by a constant rate. The promoter sequence will be repressed again while the concentration of the lactose is getting low enough. That is, to fulfill the timer function, we can control the reaction time by controlling the amount of lactose.

(3)	Output We use the green fluorescence protein (GFP) (<a href="http://partsregistry.org/Part:BBa_K145015" style="text-decoration:none;">BBa_K145015</a>) and the red fluorescence protein (RFP) (<a href="http://partsregistry.org/Part:BBa_K145150" style="text-decoration:none;">BBa_K145150</a>)as the output signal. The green indicates “on”, whereas red indicates “off”. We insert a part of sequence which encodes the GFP downstream the LacI-repressed promoter (<a href="http://partsregistry.org/Part:BBa_R0011" style="text-decoration:none;">BBa_R0011</a>). When switched on, the promoter starts translating and directly lead to the appearance of the green color. Moreover, the promoter would indirectly lead to the appearance of the red color. The timer and the counter share the same mechanism to output the signal. (4) Memory System The basic idea of this memory system was first developed by <a href="http://2008.igem.org/Team:KULeuven" style="text-decoration:none;">KULeuven</a> during the 2008 iGEM competition. It is composed of two conjugated sequences. (See more details <a href="http://2008.igem.org/Team:KULeuven/Project/Memory" style="text-decoration:none;">here</a>) <a href="http://2008.igem.org/Image:Alt2.png"> <img src="http://2008.igem.org/wiki/images/5/5b/Alt2.png" width="500" height="234" border="0" > </a> By the work of these sequences, the inducer (lactose) is distinguished and memorized, and then drives the whole device. When the inducer is removed, however, the function of whole device can be switched to a contrary one. ( It can only be switched on once and can never go back. ) We use the lactose to control the initiation and closure of the system. However, it’s contradictory that the system should initiate the counter only in the final stage but not in the second stage; even the functions in these two stages are both shut down. Hence, we can save the input signal into the memory system to solve the problem. The memory system can remember the input signal even when external conditions have changed. In the memory system, we connect two sequences of tetR and CIIP22 proteins downstream the promoter which is repressed by the CI434 protein. During the lactose-accession stage, the translation of tetR and CIIP22 is repressed, and meanwhile promote the other sequence to produce the CI434 protein. It means that the repression of two conjugated sequences reverses. Thereafter, no matter what surrounding becomes, the product of this memory system doesn’t change.

(5) LuxR Operon Acyl-homoserine lactone (AHL) is a kind of auto-inducer. It serves as the communicative signal in the bacterial self-illumination mechanism <a href="http://2009.igem.org/Image:Lux1.png"> <img src="http://2009.igem.org/wiki/images/2/2d/Lux1.png" width="531" height="268" border="0" > </a> The LuxR protein is a membrane receptor which distinguishes AHL compound liberated by bacteria and passes down the signals. When external bacteria aggregate, the concentration of AHL will rise to the boundary level and cause the production of AHL-LuxR complex. This complex then becomes a translated factor and starts the translation of the Lux operon. Based on the Lux auto-inducer mechanism, we can insert the target sequences into the Lux operon. By doing so, we can regulate any device we want. In addition, we inserted a sequence of LuxI protein downstream of the Lux operon. The LuxI protein impels the S-adenosylmethionine (SAM) to transform into AHL. This becomes another way of regulation. (6)	The Regulation of the RFP The ultimate purpose is to reveal the red color only under the condition that the time is up or the bacteria invasion is higher than standard value. <a href="http://2009.igem.org/Image:Regulaterfp.png"> <img src="http://2009.igem.org/wiki/images/5/5c/Regulaterfp.png" width="400" height="350" border="0" > </a> First, the promoter in this circuit is a double-regulated promoter, and it can only be activated when the following requirements are fulfilled: (1) The lack of the CIIP22 protein and (2) The linkage to the AHL-LuxR complex. Hence, we can regulate the device at the appropriate timing. After the function of the memory system is shut down and the AHL-LuxR complex accumulation caused by the cease of tetR translation, the promoter (BBa_K145015) starts to work and produce the RFP. (7)	Counter The counter is designed to work in the lactose-accession stage only. We insert a part of sequence which encodes the LuxR protein downstream the constitutive promoter (<a href="http://partsregistry.org/Part:BBa_J23106" style="text-decoration:none;">BBa_J23106</a>). Therefore the system is full of LuxR on the go. Besides, the promoter in the Lux operon (<a href="http://partsregistry.org/Part:BBa_K145150" style="text-decoration:none;">BBa_K145150</a>) can only work in the lactose-accession stage under the regulation of the LacI-repressive promoter. We can use both characteristics to prompt the work of the signal output and the cell’s death. 我們的首要目的，是使大腸桿菌具有「自動計時提示」及「自動偵測細菌濃度」的功能. 啟動功能後，細菌會表現出綠色；當經過特定時間後，或外來生菌濃度過高時，大腸桿菌會轉變成紅色，以達到示警的作用. 作用過程可大略分為三個階段：(1)培養期，此時E.coli只會生長；(2)作用期，此時加入乳糖誘導物，啟動計時功能與偵菌功能；(3)警示期，當乳糖降解或外來菌過多時，啟動警示系統與自殺機制. 整個project中所用到的各部件，將在下列分開說明： (1)	開關與輸入 我們利用乳糖操縱組的原理作為整個裝置的開關，並利用乳糖作為控制開關開啟的輸入訊號. 首先，利用一個會被LacI蛋白抑制(LacI-repressed promoter,R0011)的啟動子作為整個裝置的起點，所有後續的蛋白都是由它轉譯出，並直接或間接受到它的調控. 其次，利用一個強啟動子 (J23106) 不斷的轉譯出LacI蛋白，當環境中的LacI蛋白濃度高時，容易與R0011上的位點結合並抑制其作用 (培養期). 當加入定量的乳糖誘導物後，乳糖與R0011爭奪LacI的結合位，使結合在R0011位點上的LacI逐漸減少，也開啟了後續反應的進行 (作用期). 當乳糖逐漸被降解時，LacI對R0011的結合又再佔優勢，也再次使反應轉換 (警示期). 簡單的說，加入乳糖即可啟動整個系統，而缺乏乳糖時系統再度關閉. (2)	計時功能 在養菌期時系統是關閉狀態；當外加定量乳糖時，開關會被開啟；而乳糖濃度會因降解而慢慢變低至殆盡，此時開關將會再度關閉. 因此，只要開啟及關閉時輸出不同訊號，我們將可以得知乳糖降解的這段時間長度；而這段降解時間可藉由改變乳糖的初濃度控制，進而得到計時功能. (3)	輸出訊號 我們以綠螢光蛋白及紅螢光蛋白做訊號輸出：綠色表示開啟，而紅色表示關閉. 我們在LacI-repressed 啟動子 (R0011) 後方直接接上綠螢光蛋白的基因序列，當開關開啟時，啟動子被啟動，並直接使綠螢光蛋白表現. 另外，由LacI-repressed啟動子間接控制紅螢光蛋白的轉譯. 其中，計時功能與偵菌功能將共用此輸出方式. (4)	記憶系統 記憶系統是由魯汶大學在2008iGEM中提出的套件，是由兩段共軛的基因序列組成，(詳細原理請見2008KULeuven http://2008.igem.org/Team:KULeuven/Project/Memory). 加入的誘導物(果糖)可調控共軛的兩條序列，進而達到我們所需求的功能. 如果移除誘導物，則能將整個系統切換成另一個狀態並產生另一種功能. (這組共軛序列只能進行一次切換，而且這切換是不可逆的. ) 在我們的作品裡，利用乳糖的有無控制系統的開與關，然而，系統在培養期與警示期時都同樣處於關閉的狀態，卻又要在警示期產生偵菌效果，這是一個比較矛盾的差異所在. 此時，就可以將作用期輸入的指令儲存在這套記憶系統內，當環境改變時，系統仍能儲存原有指令，以達成兩個相同狀態下所須輸出的差異. 我們在記憶系統中，其中一條基因序列下游加上tetR與CIIP22兩種蛋白編碼序列，而其上游的啟動子會被CI434蛋白所抑制. 培養期時，tetR與CIIP22不斷被轉譯出並調控後續元件；作用期時，乳糖間接促進CI434蛋白的轉譯並影響記憶系統，使tetR及CIIP22的轉譯被抑制，與此同時也間接促使其共軛序列轉譯出CI434蛋白. 此後，無論外界誘導物濃度或整體反應環境如何變化，tetR及CIIP22將始終受到CI434抑制而達成記憶的作用. (5)	LuxI、LuxR作用系統 Acyl-homoserine lactone (AHL) 是一種Autoinducer，在發光菌的發光機制中起著傳遞訊號的作用. 發光菌藉由這個機制，使其本身對其細胞密度有加以監控之能力. 在臨界濃度以上，AHL便會與進入另一個細胞體內與基因產物LuxR 結合，LuxR進一步作為轉錄因子，細菌就會大量的轉錄Lux operon中之基因. 我們便可利用這個機制的原理，在LuxR operon中加入欲受調控的基因序列. 另外，LuxI會促使S-腺?甲琉胺酸 (SAM) 轉變為AHL，成為另一個調控Lux operon的方法. (6) 紅螢光蛋白的控制 這是整個裝置中最複雜的部份. 我們調控的最終目標為，紅螢光蛋白只有在計時器計時完畢，或是外來菌濃度過高時表現. 由於在培養期與計時期都不予表現，所以在設計上經過一番巧思. 首先，RFP上游的K145150是一個受雙重控制的啟動子，要啟動它必須同時滿足：(1)環境中沒有CIIP22蛋白及 (2)與AHL-LuxR複合體結合，兩種條件. 因此，我們就可以在適當的時機去控制其運轉：當記憶系統的轉譯功能被關閉 (加入乳糖啟動裝置的階段，培養期)，以及tetR不再被轉譯進而造成AHL-LuxR複合體累積 (乳糖逐步被分解，作用期) 後，K145015開使運轉，造成紅螢光蛋白的表現並達到警示的目的. (7)	偵菌系統 我們希望在偵菌的功能只在作用期能夠作用，因此在強表現啟動子的下游插入一段 LuxR蛋白的編碼序列，使系統中隨時都存在LuxR蛋白；另外Lux operon中的啟動子K145150只有在作用期時不被抑制. 只要利用Lux作用系統的原理：當外來菌AHL過高時，AHL-LuxR複合體便可啟動K145150，並進一步造成紅螢光警示與細胞凋亡.