Tianjin/DIODE/Experiment1

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Revision as of 05:03, 26 October 2007

Introduction

As we want to get the signal AHL go throw these three cells but the cells do not mix,we decide to immobilize the generator cell(BBa_I13202),amplifier cells(BBa_I15030),block cells(BBa_I13208) using alginate sodium. Another aim of immobilize the amplifier cell is that we want to conreol the positive feedback which acted as Quorum Sensing in nature by input AHL molecular to cause a input decided system from a time decided QS system.
After immobilizing these cells ,we test them by getting the culture medium of the tested cells at different time and putting it together with detector cell for 1.5h to measure the AHL.All the fluorescence value is measured by Virian Cary Eclipse fluorescence spectrophotometer using the 3ml box at condition : Excitation wavelength-501nm(5nm),Emission wavelength-511nm(5nm),Average time 0.5s.
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Test result

Figure 1: AHL tends to be degrated gradually by immobilized block cells as both figure indicated, after 7 hours, the concentration of AHL has dropped down to less than half of the original value. The difference between the time needed to observe dramatical decline is because the number of immobilized beads on the left figure overnumbered that on the right. Therefore, block cells still work well after being immobilized.

Figure 2: To test the validity of our data, the background expression of detector cells and standard deviation of our data are also considered. As the figures show, the leakage expression of GFP is far below the level of activated expression and influent a little on final curves and the tendency of curves would not change within the range of standard deviation.

Figure 1: The remarkable increasement of AHL prove the function of immobilized generator cells which secrete AHL continually to the culture flux.

Figure 1: Following two figures are selected from five parallel experiments aiming at verifying the function of immobilized amplified cells. The function curves of generator cells and block cells are also displayed in the same figure to emphasize the effects of amplified cells. Actually, the curves of amplified cell( marked as 8P) is identical with that of generator cells, a phenomenan which could be attribute to self-activation of amplified cells because of its leakage expression of AHL. Since the threshold of Quorum Sensing is far below than our expectation, further adjustment need to make to eliminate the leakage expression of AHL to a reasonal level to avoid such a condition.




Figure 2: One of our strategy is to cut down the functional part from original plasmid and transfer into a plasmid with a copy number less than 10 without the addition of IPTG (pSB2K3). With the addition of IPTG,the copy number of this plasmid could rise to as much as more than 100, so enhancement of the amplification could be achieved. Although the result is not ideal as that of other types of cells, the graph equally shows AHL signal could be slightly magnified after the addition of IPTG and AHL. The background expression of AHL still stays at a high level which makes the curve of control identical to those added with IPTG and AHL. However, compareing the sapphirine curve with the blue curve, we can see after two hours, the AHL signal is remarkablly magnified after the addition of IPTG and AHL. The right graph substantiate our hypothesis either where the green and blue curve represent the cells with and without induction of IPTG respectively.