Team:NTU-Singapore/Wetlab/Experimental Results

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<div id="maincontent" style="margin-top:250px;">
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=Results=
 
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==Characterization of plsrA-YFP==
 
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[[Image:Yfp-time.JPG]]<br>
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='''Verification of Detection & Lysis system'''=
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As stated before, we wish to verify that the cells with the pLsrA-Lysis system would lyse upon Ai-2 inoculation. We used the OD of the cells as a form of verification in this experiment.
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For all the samples with AI-2 added, relative fluorescence unit (RFU) increases with time. In contrast, the cell samples with 50 µl water added have their RFU low and not varied much. The control samples with addition of 50 µl of LB have higher RFU level, yet it remained steady with time. From this observation, it can be concluded that the increase in RFU of the samples with AI-2 addition is not related to cell growth as as result of LB presence in the supernatants. Hence, this increasing trend can be well explained as YFP was actually expressed in the presence of AI-2.
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=='''OD600 measurement of pLsrA-Lysis-containing LuxS mutant upon addition of AI-2'''==
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This further proves that the LsrA promoter part BBa_K117002 in fact works correctly as it is activated in the presence of AI-2. Further characterization of this promoter will be implemented in future works.
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From this result, it is highly expected that the detection system part BBa_K117010 also expresses lysis protein (celE7) upon induced by AI-2.
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[[Image:Ai2_on_YFP.JPG]]<br>
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[[Image:OD time.png|thumb|center|800px|Changing OD]]<br/>
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This clustered column graph shows a clearer view on RFU expressed by different samples throughout the experiment. As expected, RFU levels of the control samples (cell suspension with addition of water and LB) are very low and not varied much compared to the high and increasing RFU of the samples with AI-2-containing supernatants.   
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For this experiment, samples of LuxS mutants carrying pLsrA-lysis plasmid were introduced with supernatants containing AI-2 and the optical density was measured. As pure AI-2 are very hard to obtain, we harvested them indirectly by taking the supernatants of the wildtype bacteria after centrifuged and filtering through 0.2µm pore size filters (refer to [https://2008.igem.org/Team:NTU-Singapore/Wetlab/Materials_and_Equipment Materials and Equipment] for more details). So the supernatants that we used for testing do not contain only AI-2 but also LB broth. Therefore, for all our testing experiments, we used bacteria added with LB as negative control.  <br/>
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Another conclusion can be drawn is that AI-2 activity seems to be highest for the supernatants obtained after 8 hours of incubation.
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We can observe that initially, the curves of cell samples with LB and AI-2 have slightly higher slopes than that of sample with cells only. This can be easily understood because the supernatants also contain considerable amounts of LB, which promotes cell growth.<br/>
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However, after about 3 hours, the cell induced with AI-2 reached steady state while the control samples were still growing. This can be well explained by the effect of lysis, which counters cell growth at the same time.
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Therefore, the cells in fact lyzed under the induction by AI-2. Or in other words, '''our detection system works as expected!!'''
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<br><br>
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<html>
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==OD600 measurement of pLsrA-Lysis-containing LuxS mutant upon addition of AI-2==
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===Initial Phase===
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The following picture shows when AI-2 solution is added at initial time
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[[Image:OD_vs_AI2.JPG]]<br>
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Note that, cell only means there is only 200 µL cell in the well, AI-2, 2 means the AI-2 solution is collected at 2hrs.
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The results shows that with LB or water the cell culture reach higher steady state comparing to that with AI-2 solution as we can see the divergence at around 4hrs. all AI-2 solution (AI-2 3, 4, 5, 6, 8) except AI-2, 2 shows depressed steady state, indicating that, AI-2 is actually working
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===After 1 hr===
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The following picture shows when AI-2 solution is added at 1hr later
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[[Image:OD_vs_AI2_1hour.JPG]]<br>
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This also shows the expected result, with AI-2 present, the cell culture shows a lower steady state.
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[[Image:AI2_on_LsrA-Lysis.JPG]]<br>
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This error bar graph shows OD600 of each sample at different time intervals together with the standard deviation. It is clear from the graph that the variance of data is much smaller than the absolute value. Hence, the data obtained from the absorbance-meter is highly reliable.
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Latest revision as of 23:54, 28 October 2008



Verification of Detection & Lysis system

As stated before, we wish to verify that the cells with the pLsrA-Lysis system would lyse upon Ai-2 inoculation. We used the OD of the cells as a form of verification in this experiment.

OD600 measurement of pLsrA-Lysis-containing LuxS mutant upon addition of AI-2

Changing OD

For this experiment, samples of LuxS mutants carrying pLsrA-lysis plasmid were introduced with supernatants containing AI-2 and the optical density was measured. As pure AI-2 are very hard to obtain, we harvested them indirectly by taking the supernatants of the wildtype bacteria after centrifuged and filtering through 0.2µm pore size filters (refer to Materials and Equipment for more details). So the supernatants that we used for testing do not contain only AI-2 but also LB broth. Therefore, for all our testing experiments, we used bacteria added with LB as negative control.
We can observe that initially, the curves of cell samples with LB and AI-2 have slightly higher slopes than that of sample with cells only. This can be easily understood because the supernatants also contain considerable amounts of LB, which promotes cell growth.
However, after about 3 hours, the cell induced with AI-2 reached steady state while the control samples were still growing. This can be well explained by the effect of lysis, which counters cell growth at the same time. Therefore, the cells in fact lyzed under the induction by AI-2. Or in other words, our detection system works as expected!!

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