Team:NCTU Formosa/WetLab/Memory





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Result - Memory System

Outline The basic idea of this memory system was first developed by KULeuven during the 2008 iGEM competition. This memory system is applied in our referee system to memorize that the inducer (lactose) has been added or not. When the inducer is removed, the function of whole device can be switched to a contrary one. (It can only be switched on once and can never go back). Therefore, the memory system maintains media colorless until the lactose is added. <a href="http://2009.igem.org/Image:Timer_new01.png" ><img src="http://2009.igem.org/wiki/images/4/44/Timer_new01.png" border="0"></a> Component Descriptions

Strand A   	<ol> <li>PcI434 (BBa_R0052) is inhibited by cI434-LVA(BBa_C0052) protein .</li> <li>The promoter PcIIP22 (BBa_R0053) is inhibited by cIIP22 (BBa_C0053) protein.</li> <li>Without any control, the promoter PcI434 is stronger than the promoter PcIIP22 in transcription.</li> <li>RBS (BBa_B0031) on strand D have a better efficiency of transcription than the RBS (BBa_B0033) on strand E.</li> <li>The promoter Plac (BBa_K091110) is inhibited by lacI (BBa_K091121) protein.</li> </ol>

<Strong>Principle and Mechanism</Strong> The memory system in our bacterial referee has three stages: <ol> <li>Standby phase: bacteria grow in lactose-free medium.</li> <li> Lactose-accession phase: lactose is added in medium. </li> <li> Lactose-consumption phase: bacteria eat all lactose, and the medium does not contain lactose. </li> </ol> <ol> <li>Standby stage: the medium is colorless <a href="http://2009.igem.org/Image:Memory02.png" ><img src="http://2009.igem.org/wiki/images/d/d8/Memory02.png" border="0"></a> When bacteria grow in the lactose-free medium, the lacI (BBa_K091121) protein is produced by constitutive promoter and represses the Plac (BBa_K091110) promoter. Therefore, green fluorescent protein with LVA tags (GFP-LVA) (BBa_K145015) is not expressed in the medium. Since the transcription activity of the PcI434 promoter is stronger than the PcIIP22 (BBa_R0053) promoter, cIIP22 (BBa_C0053) can be produced and repress the PcIIP22 promoter and the Plux/cIIp22 (BBa_K145150) promoter. The Red Fluorescent Protein (RFP) ( BBa_E1010) is also not expressed in the medium. The medium is colorless in this stage. </li> <li>Lactose-accession phase: the medium is green <a href="http://2009.igem.org/Image:Memory03.png" ><img src="http://2009.igem.org/wiki/images/9/9f/Memory03.png" border="0"></a> When lactose is added in medium, lactose binds with lacI (BBa_K091121) repressor and releases the inhibition of B strand, so the PlacI (K091110) promoter starts transcripting cI434-LVA(BBa_C0052) proteins and GFP-LVA(BBa_K145015). Then, the medium are green in this stage and the cI434 repressor inhibits the PcI434 (BBa_R0052) promoter. E strand stops the inhibition to the promoter PcIIP22 (BBa_R0053) of D strand, so the promoter PcIIP22 starts to slowly transcripting cI434-LVA (BBa_C0052)protein to inhibit the promoter PcI434 (BBa_R0056)of E strand. Because transcription effects of D strand are not strong, and the repressor protein cI434-LVA has relatively high degradation rate, it is difficult to inhibit E strand by itself, but fortunately the B strand cI434 proteins provide a another force of strong and lasting inhibition that can help D strand repress of the promoter PcI434 of E strand. </li> <Br> <li>Lactose-consumption phase: the medium is red <a href="http://2009.igem.org/Image:Memory04.png" ><img src="http://2009.igem.org/wiki/images/6/6c/Memory04.png" border="0"></a> When bacteria eat all lactose, lacI (BBa_K091121) repressors start inhibiting the promoter Plac again. B strand stops transcription of cI434 proteins, and D strand has been transcribed a considerable number of cI434-LVA (BBa_C0052) protein in the second stage. The Promoter of PcI434 (BBa_R0052) is still suppressed at this stage. In this stage only the A, D and F strands are working. Therefore, the RFP ( BBa_E1010) is expressed by the Plux/cIIp22 (BBa_K145150) promoter, the medium is red in this stage. </li> </ol>

Important concept Have you noticed? There is no lactose in the first stage and third stage; however, the working DNA strands are different. That is the memory device record" lactose had been added" message in our bacterial referee system. Submitted parts in this memory

<a href="http://2009.igem.org/Image:Memory05.png" ><img src="http://2009.igem.org/wiki/images/5/59/Memory05.png" border="0"></a>