Team:NCTU Formosa/WetLab/Group3

Principle

The basic idea of this memory system was first developed by KULeuven during the 2008 iGEM competition. Now we serve it as a function to memorize the added inducer. By conjugating the 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. )

記憶系統的基本構想是由KULeuven於iGEM2008所提出，而我們將他延伸運用在「被加入的誘導物與環境控制的記憶」此功能上。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。)

The four devices above were separated from our project and be interpreted individually. T7 promoter (I7 17024) is a strong, continually expressed promoter, so that the LacI gene sequence (K091121) is translated unceasingly and lead to the accumulation of the LacI protein. Surrounded by LacI protein, the SD sequence of the PLacI promoter (K091110) is easily stocked and restrains the promoter’s work.

本小組將以上四組devices獨立出來測試與說明。
T7是一個強啟動子，所以LacI 會持續不斷的被轉譯出並累積，抑制住PLacI promoter。在充滿著LacI的環境下，啟動子的SD序列會被包覆並阻礙轉錄及轉譯的進行。

Devices C and D are two conjugated sequences and they repress each other. Pci434 is a strongly expressed promoter and more powerful than promoter Pciip22, so device C is repressed at the beginning. When the activator is added, however, CI 434 protein is produced and accumulated. Therefore, the repression reverses. Once device C expresses, device D will be repressed till the end. No matter what surrounding becomes, the product of this memory system doesn’t change. That is the function what we called “memory”.

C和D兩段序列是互相抑制的共軛序列；然而，位於D序列上的啟動子Pci434較C序列上的啟動子Pciip22為強，故在反應起始時，D序列的表現作用較強而會抑制C序列。在加入誘導物後，B序列轉譯出CI434蛋白並抑制Pci434，導致抑制方向逆轉。一旦C序列表現強於D序列，D序列就會被永遠抑制住，此時不論外在環境，此共軛片段產生出之結果都不再改變，也達成了「記憶」的效果。

More detail about the conjugated sequences is researched and published by KULeuven in 2008. See here .

Experiment

KULeuven have had already published the memory system in 2008 and built its model (see here), but the construction of the whole device was left undone. So the core work in our group is to construct the memory system and experiment with the relating data. First we add the DNA sequence of GFP with LVA-tag into device D. If the green fluorescence was detected, we know that the device works. On the other hand, if the conjugated function works, the transcription of GFP-LVA sequence will soon shut down and the signal of the green fluorescence will reduce, by which can we ensure the effectiveness of the system.

魯汶大學在2008年的iGEM競賽中即發表過這套記憶系統 (see here)，也建立了相關的數學模型，但實際上尚未成功的接出整組序列。所以本小組的首要任務，就是將這套記憶系統序列建構出來，並實際測量反應量、反應時間等相關數據。
我們的計畫是：在其中一條序列下游，另外加上一段可轉譯出綠螢光蛋白的序列，如此，在尚未打開LacI開關時，大腸桿菌會發出綠螢光；當LacI開關被打開時，共軛序列被切換，不再轉譯出綠螢光蛋白。因此，綠螢光蛋白會自然降解，我們可藉由偵測綠螢光的吸光度來斷定此二共軛序列是否如理論般作用。

After the construction, we are going to test the concentration of the input lactose and the fluorescent signal output and test the POPs. We will also exam this system under different circumstances to ensure the system works under the demanded conditions.

如果記憶系統建造成功，我們將進一步測定誘導物 (lactose) 和綠螢光吸光值的關係，做出POPs；同時在各種不同的環境下作測試，以求此系統能在特定需求的環境下作用。