Team:PKU Beijing/Parts Characterization/BBa K228004/Growth

Parts > Parts Characterization > BBa_K228004 > Growth Rate

BBa K228004 - NahR( reverse) - salicylate promoter
Designed by Lin Min  Group: iGEM09_PKU_Beijing   (2009-09-18) Input: Salicylate molecules Output: GFP fluorescence

Description
It has been observed that salicylate as a inducer may decrease the growth rate of E. coli, but what the pattern and level of this decrease is unknown to us. We characterized the growth rate of E. coli through the method described in the Part Main Page. We processed the data as the protocol mentioned in the Transfer Function page, demonstrating that the decrease level is significantly and positively correlated with the concentration of salicylate.

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Figure 1 - The relationship between growth rate and concentration of the inducer. We detected cell numbers in 20mins, 40mins, 60mins, 80mins, 100mins and 120mins, presented by corresponding points in this figure. Error bars shows the upper and lower limit of confidential interval. These curves reveal the decrease on growth rate in the presence of the inducer, salicylate molecule. We observed that at each time point of 20mins, 40mins, 60mins, 80mins, 100mins and 120mins, the decrease level is significantly and positively correlated with the input, the concentration of salicylate.======

Protocol
1. We characterized the growth rate of E. coli through the same method as described in the Transfer Function page. The time between repeated samplings is 20 minutes. As the same, 6 replicates were measured for each concentration of salicylate, and the concentration 0 group is to measure fluorescent background.

2. We used spectrophotometer to test the OD600 value of the left 100 ul of bacteria resuspention. Then the OD600 values were conversed into the concentration of cells (/ml). The equation is OD600 1.0=5*10^8 cells/ml.

3. The curves in Figure 1 show the relationship between growth rate and concentration of the inducer. We detected cell numbers in 20mins, 40mins, 60mins, 80mins, 100mins and 120mins, presented by corresponding points in the Figure 1. Error bars shows the upper and lower limit of confidential interval. These curves reveal the decrease on growth rate in the presence of the inducer, salicylate molecule. We observed that at each time point of 20mins, 40mins, 60mins, 80mins, 100mins and 120mins, the decrease level is significantly and positively correlated with the concentration of salicylate as expected.

4. But we also note that there are several abnormal points on these curves. We propose that these abnormal points are caused by the noise from the induction system itself, as in our other similar experiments abnormal points occurred randomly.

5. It has been observed that salicylate as an inducer may decrease the growth rate of E. coli, but the mechanism beneath this decrease on growth rate has not been characterized very convincingly. Reassuringly, we observed that arabinose, a small molecule which can act as the carbon source for E. coli, can also trigger the decrease on growth rate in our another test system designed for BBa_K228009. It was not expected that, however, the inhibition resulted from arabinose can be lifted two hours later after the inducer added, and the finatl concentrations of cells/OD600 values after two hours have no significant differences. In addition, we found that the presence of this disinhibition is universal in the chemical induction of arabinose. Accordingly, we propose that arabinose, which can act as a carbon source, will be consumed in metabolism culminating in a disinhibition of growth rate, while salicylate, which can not be catabolized, will maintain a nearly constant concentration in the cytoplasm, eventually resulting in a sustained inhibition and thus decrease the growth rate.