Team:NTU-Singapore/Wetlab/Experimental Results

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Contents

Results

Characterization of plsrA-YFP

Yfp-time.JPG

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. 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. From this result, it is highly expected that the detection system part BBa_K117010 also expresses lysis protein (celE7) upon induced by AI-2.

Ai2 on YFP.JPG

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. Another conclusion can be drawn is that AI-2 activity seems to be highest for the supernatants obtained after 8 hours of incubation.

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

Initial Phase

The following picture shows when AI-2 solution is added at initial time OD vs AI2.JPG

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. 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

After 1 hr

The following picture shows when AI-2 solution is added at 1hr later OD vs AI2 1hour.JPG

This also shows the expected result, with AI-2 present, the cell culture shows a lower steady state.

AI2 on LsrA-Lysis.JPG

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.