Team:NCTU Formosa/Project/OverView

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OverView In order to produce a timer function in our system, we design the regulation  devices between LacI,  LacI promoter and lactose. We control the devices by adding  lactose to activate  our constructions. The following is the first device:

為了使我們的系統具有計時功能，設計調節的功能在LacI,、LacI promoter和lactose之間，以外加lactose的方式來調節和促進此基因序列的表現. 以下是第一個設計的裝置 : The First Device

<img width="415" height="113" src="project_clip_image002_0000.png" />

<img width="432" height="137" src="project_clip_image003_0000.png" align="left" hspace="5" alt="文字方塊: Name	Number	Well	Length	Resistance	Plasmid Constitutive promoter	BBa_J23106	P1-18O	35bp	A	BBa_J61002  RBS	BBa_B0034	P1-2M	12bp	A	pSB1A2  Lac I	BBa_C0012	P1-2O	1128bp	A	pSB1A2  Lux R	BBa_C0062	P1-4O	756bp	A	pSB1A2  Double Terminators	BBa_B0014	P2-24C	95bp	AK	pSB1AK3v     " > Total:2024bp To inhibit transcription of the LacI promoter (pLacl, BBa_R0010), we have to encode LacI protein (BBa_C0012). LacI protein can bind to pLacI and repress the expression, but lactose, which repress lacI  binding, act as a switch opening the expression. Therefore, the inter-regulation could be a timer device, so LacI is a key component of first device. The second gene is the LuxR, which is a membrane receptor,  takes external AHL compound to pass down the  signals. We use the LuxR as our bacteria-sensor to sense other bacteria in the environment. For selecting other parts, we choose one kind of constitutive promoter family(BBa_J23106) as the promoter, and the expression amount of this promoter is moderate  comparing others in the family. Furthermore, we use the commom ribosome-binding site(BBa_B0034) and  the terminator(BBa_B0014), which is a special double terminator, to  ensure the transcription would exactly stop.

為抑制LacI promoter(pLacl, BBa_R0010)的開啟，我們在此裝置插入LacI 蛋白質 (BBa_C0012)的編碼使LacI 蛋白質能與pLacl結合，抑制pLacl開啟後段基因的表現，但為調控此裝置，我們又設計lactose作為一個開關，它能抑制LacI 蛋白質與pLacl的結合，進而又開啟下游基因的表現，因此，這種內嵌式的調控可作為一個計時機制，使LacI成為此裝置的關鍵因子. 第二個基因LuxR是細胞膜上接受外來AHL複合體的受體蛋白質，可傳遞訊息到細胞內，經由接收AHL複合體可以用來偵測外在是否有菌汙染環境，而此裝置的promoter在constitutive promoter family(BBa_J23106)中選擇較溫和表現的來使用，另外，也選擇普遍使用的ribosome-binding site(BBa_B0034)和terminator(BBa_B0014)雙停止子來確保轉譯確實停止. The Sixth Device:

<img width="415" height="116" src="project_clip_image002_0001.png" />

Total:868bp In the final device in our sequence, we encode the promoter (BBa_K145150), which can be induced by the LuxR-AHL complex, and inhibited by protein cIIp22 (produced in previous device). When LuxI is produced ( produced when lactose consumed in previous device), it can  stimulate the formation of AHL, which can be regarded as outer bacteria invasion. Therefore, the LuxR-AHL complex can be used as signs of timer and detecting outer bacteria. Finally, the gene mRFP1(BBa_E1010) is encoded, which can illustrate red fluorescence, let we  know that the time is up or outer bacteria have invaded. Furthermore, we use the common RBS(BBa_B0034) and the double terminator(BBa_B0014) to ensure the transcription  would exactly stop. In our experiments, we only encode mRFP1 without gene ccdB, because we only interest in how the promoter and gene mRFP1 demonstrate. Find out the appropriate concentration of LuxR –AHL complex that induce the promoter and the degree of the red fluorescence intensity.

在第六個裝置中，我們選用可被LuxR-AHL 複合體誘發而表現，並且被cIIp22 蛋白質 (產生在第五個裝置中)所抑制的promoter (BBa_K145150)，當LuxI產生時會促使AHL的形成，可視為外來菌侵入的標記，所以LuxR-AHL 複合體可以被用來作為偵測到外來菌的訊息. 另外，插入一段mRFP1(BBa_E1010) 基因可以表現紅螢光，當菌發出紅色的訊息時，我們就知道計時時間已到，以及有過高的外來菌侵入. 此外，也選擇普遍使用的ribosome-binding site(BBa_B0034)和terminator(BBa_B0014)雙停止子來確保轉譯確實停止. 在此裝置的實驗中，因只確認promoter和mRFP1是否作用，以及找出合適的LuxR –AHL 複合體濃度以誘發promoter開啟，mRFP1紅螢光的強度測試，並沒有插入ccdB基因.