Measurement
pXS-Nic2a

Part:BBa_K2827005

Designed by: iGEM18_Worldshaper-XSHS   Group: iGEM18_Worldshaper-XSHS   (2018-10-07)


nic A2 promoter+GFP

It's a nicotine sensor with GFP reporter, which can be activated when encountering nicotine limitation.

Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI.rc site found at 707


Usage and Biology

Part BBa_K2827005 is the first version of nicotine detector designed by Worldshaper-XSHS. NicA is an important enzyme involved in nicotine degradation by Pseudomonas putida strain S16, which can convert nicotine to N-Methylmyosmine. The promoter activity of nicA gene may be related to the concentration of nicotine. In part BBa_K2827005, we added a reporter gene after the nicA2 promoter. The nicA2 promoter is induced by nicotine. When the promoter is activated, the GFP expresses.

Host organism

This plasmid is used in E.coli DH5α.

Functional analysis

Detection of GFP expression

1.Transform part BBa_K2827005 into competent cells(E. coli DH5α)

2.Allow the bacteria to grow overnight in LB agar plates with or without nicotine(5g/L)

3.After overnight cultivation, observe the E. coli DH5α cells with a fluorescence microscope.

Result

When the bacteria grew in LB plate without nicotine, no GFP expression was observed(fig.1). While the bacteria were cultivated in LB agar plates with nicotine(5g/L), GFP expression was discovered(fig.2). The transcriptional activity can be induced by nicotine.

5-1.jpeg

fig.1 the GFP expression in LB plate without nicotine

Left: under the white light; Right: under the blue light

5-2.jpeg

fig.2 the GFP expression in LB plate with nicotine

Left: under the white light; Right: under the blue light


sensitivity of the detection system

Activation of transformed E.coli DH5α

1.Add 30µL E.coli DH5α (transformed with BBa_K2827005) to 3ml LB and incubate overnight.

2.Add 90µL E.coli DH5α(from step1,OD600=0.8) to 3mL LB and incubate for 5h.

3.Add 30µL E.coli DH5α(from step2,OD600=0.4) to 3mL LB and incubate for 14h.

4.Add 90µL E.coli DH5α(from step3,OD600=1.3) to 3mL LB and incubate for 5h. After 5h’s incubation, the OD600 would be about 0.4.

Determination of the optimum concentration for bacterial growth

1. Prepare nicotine solutions of different concentrations in LB: 0、0.02、0.04、0.08、0.16、0.32、0.64、1.28、2.56、5.12、10.24 (g/L)

2. Add 3µL E.coli DH5α(OD600=0.4, transformed with BBa_K2827005) to 100µL LB in a 96 well plate.

3. Measure the OD600 value and fluorescence intensity value every two hours by a plate reader.

Results

High concentrations (above 1.28g/L) of nicotine had a strong inhibitory effect on the growth of E.coli, which affected the expression of GFP in cells(fig.3), so we reduce the range of nicotine concentration and carry out a second measurement.

5-3.jpeg

fig.3 the inhibitory effect of nicotine on bacterial growth

The second measurement

1. Prepare nicotine solutions of different concentrations in LB: 0、0.001、0.01、0.1、1 (g/L)

2. Add 3µL E.coli DH5α(OD600=0.4, transformed with BBa_K2827005) to 100µL LB in a 96 well plate.

3. Measure the OD600 value and fluorescence intensity value every hour by a plate reader.

Results

Within 4 hours, the bacteria growing in LB with nicotine( 0.001g/L and 0.01g/L) showed much stronger GFP expression than the bacteria growing in LB without nicotine, and the fluorescence intensity increased steadily. While the concentration of nicotine was C=0.1g/L or C=1g/L, the fluorescence intensity was lower than the control group, which is negative in fig.4.

5-4.jpeg

fig.4 the fluorescence intensity of E.coli DH5αin different conditions

model

(for more details: [http://2018.igem.org/Team:Worldshaper-XSHS/Model Model] )

the correlation between bacterial growth and the concentration of nicotine

We analyzed the correlation between k (bacterial growth coefficient) and the concentration of nicotine, the results of which showed that k and pC (-lg c) had a significant positive correlation. The higher the concentration of nicotine, the smaller k will result in, that is, the stronger the inhibition of growth. The fitted function is as follows:

K-lgc.jpeg

When the concentration of nicotine is around 10^(-1.5)g/L (approximately 0.032 g/L), the growth of bacteria begins to be inhibited. As the concentration increases, the inhibition increases. When it changes to 10^(-1)g/L (0.1 g/L), the bacteria growth is inhibited to a certain degree, where k is calculated to be -3.62038186 based on the formula. When the concentration increases to 10^(-0.5)g/L (around 0.32 g/L), the inhibition is significant, and k is calculated as -17.56950279.

5-5.jpeg

The Growth Curves under Different Nicotine Concentrations

During the data analysis, we performed the following substitution of the concentration:

XN.jpeg

in order to better discover its relationship with OD600. For the X-axis, 1, 2, 3 until 10 respectively represent the 10 different concentrations of nicotine, i.e. 0.02 g/L, 0.04 g/L, 0.08 g/L, 0.16 g/L, 0.32 g/L, 0.64 g/L, 1.28 g/L, 2.56 g/L, 5.12 g/L and 10.24g/L. We can see that the OD values at 0h in different groups are all 0.05, which means that all the groups share the same starting point. At 2h, the OD values detected at various concentration groups are basically the same, and have not increased much since 0h. At 4h, the OD values of the low concentration groups increased significantly. And for the groups larger than 5 (that is, 0.32 g/L), there is a downward trend, that is, the toxicity of nicotine has begun to manifest. The critical value, the concentration of 0.32 g/L, is basically consistent with our conjecture based on the models in the first part. As time goes on, the toxicity of nicotine becomes more and more significant. At 12h, when the concentrations are greater than 0.32 g/L, the number of bacteria decreases significantly. And as the concentration increases, the inhibiting effect also increases. Based on this model, we speculate that the effect of nicotine on bacterial growth is related to time and nicotine concentration. When the exposure time is less than 4 hours and the concentration is lower than 0.32 g/L, the inhibition is not significant. While with the increase of time and nicotine concentration, its inhibitory effect gradually increases.

5-6.jpeg

[edit]
Categories
Parameters
None