Team:NCTU Formosa/WetLab/Mainworks

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WetLab: Our MainWorks Experiments

The experiment part 1 is demonstrated for finding out the time when bacteria would express the mRFP1(emit the red  fluorescence), which means the bacteria “kill” themselves, and choosing the  appropriate O.D. value to do the part 2. The part 2 is major part in this section and we take the Barry Canton’s research of PoPS receiver in 2008 for  reference. In the part 2 experiment, we culture the bacteria with  different concentrations and variants of AHL in the specific O.D. value  we get from Part1. The goal is to observe what the concentration of AHL and when the bacteria will activate its self-destruct device.

實驗分成兩個部分，part 1找出細菌從開始養到死亡發出紅螢光的確實時間，並且選擇合適進行part 2實驗的O.D.值. 而part 2的實驗，我們參考Barry Canton在2008年對PoPS receiver的研究，在固定的O.D.值培養細菌，以不同濃度和種類的AHL誘發下，引起細菌自身死亡，此目的在於測得AHL的極限濃度以及何時細菌會啟動自我死亡機制. Part1 <ol> <li>Construct #1 and #6 (without ccdB) devices into plasmids.</li> <li>Transform the assembled plasmids into the hosts, and culture it at 37°C for 16 hours or overnight. </li> <li>Pick up one of colonies, place it into LB tube,  which with 2% glycerol for nutrient, and incubate it at 37°C, and then  test growth curve. </li> <li>After 4~6 hours, test the O.D. every hour, and draw the fluorescence intensity curve until O.D. = 1. </li> <li>Find out the appropriate O.D. to do the next part.</li> </ol> <ol> <li> 在質體中建構第一個及第六個devices (沒有ccdB gene的植入) </li> <li> 將組好的質體轉入宿主中，在37°C下培養16小時或隔夜 </li> <li> 挑起plate中的一個菌落置入含有2% glycerol 做為營養源的LB tube在37°C下培養，並且測試其生長曲線 </li> <li> 培養4~6小時之後，每1小時測O.D.值，畫出螢光強度曲線直到O.D.值=1</li> <li> 找出合適做實驗的O.D.值進入part 2實驗 </li> </ol> Part2 <ol> <li>Culture the two host cells, one carries the first and sixth device and another carries only the first device. In this case, after overnight, the cultures were diluted into 20 ml of fresh medium in a 200 ml flask and shaken at 220 rpm during growth. </li> <li>Choose some of the common AHL variants and preload them  into a flat-bottom 96 well plate in eight  different final concentrations (0, 1E-10, 1E-9, 1E-8, 1E-7, 1E-6, 1E-5, and 1E-4 M). Three wells are filled with each 200 μl of media to measure the absorbance background. Three further wells are filled with 200 μl of the culture with only the first device to measure the  fluorescent background. </li> <li>Seventy-two 200 μl aliquots of the culture with the first and the sixth device were  transferred to the plate. Three replicate wells were filled for each concentration of each AHL. </li> <li>The plate was incubated at 37°C and assayed with absorbance measurements by CyFlow. The time between repeated measurements was 2 min and 21 sec. </li> <li>Repeat once the steps 1 to step 4 with three more AHL variants and again with the final two AHL variants. The time between repeated measurements was kept  fixed in each case.</li> </ol> <ol> <li>培養兩種E.coli宿主，其一為帶有第一及第六個裝置，另一種僅帶有第一個裝置. 經過隔夜培養後，將菌液稀釋至20ml的新鮮培養中，以220轉速培養. </li> <li>選擇幾種常見的AHL變異物預先以不同的終濃度(0, 1E-10, 1E-9, 1E-8, 1E-7, 1E-6, 1E-5, and 1E-4 M)放入96孔平底盤，三個孔放入200 μl的培養液以測得吸收背景值，另外三孔填入200 μl僅帶有第一個裝置的菌液以測得螢光強度的背景值. </li> <li>72管200 μl帶有第一及第六裝置的菌之菌液轉移至96孔plate上. </li> <li>整個plate放置在37度的恆溫箱下培養，並以分光光度計與流逝細胞儀每一段時間測量一次. </li> </ol>