Measurement
pXS-Nic3a

Part:BBa_K2827006

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


nicA2 promoter+RBS+T7 RNA polymerase+T7 promoter+ GFP

This part is made up of four parts: nicA2 promoter, T7RNA polymerase T7 promoter and GFP. NicA2 promoter is induced by nicotine. When the promoter is activated, T7RNA polymerase expresses. Then the T7RNA polymerase will bind to T7 promoter, which will be reported by GFP.

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 3419


Usage and Biology

This part is improved by the part BBa_K2827005, which contains a nicA promoter and a GFP. We added a T7 RNA polymerase and a T7promoter in the part BBa_K2827006, which was named version two detector. Compared with BBa_K2827005, the part BBa_K2827006 had a stronger signal generated by GFP.


Host organism

This plasmid is used in E.coli DH5α.

Functional analysis

Detection of GFP expression

1.Transform part BBa_K2827006 into competent cells(E. coli DH5α)

2.Allow the bacteria to grow overnight in LB agar plates with nicotine(5g/L)

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

Results

Compared with bacteria transformed with part BBa_K2827005, a stronger GFP expression could be observed in bacteria with part BBa_K2827006(fig.1 for part BBa_K2827005, fig.2 for part BBa_K2827006).

6-1.jpeg

fig.1 the GFP expression in bacteria with part BBa_K2827005

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

6-2.jpeg

fig.2 the GFP expression in bacteria with part BBa_K2827006

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

Comparison of the two detection systems

1. Prepare nicotine solutions of different concentions in LB: 0、0.0001、0.001、0.01 (g/L)

2. Add 3µL E.coli DH5α(OD600=0.4, transformed with BBa_K2827005 or BBa_K2827006) to 100µL LB in a 96 well plate.

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

Results

When the nicotine concentration was 0.0001g/L, the fluorescence expression of the part BBa_K2827006 was observed from 1 h,which increased steadily between 1-3 h. While the bacteria wiht the part BBa_K2827005 not showed fluorescence expression until 4h(fig.3).The results show that the version two detection system(with the part BBa_K2827006) greatly improves the sensitivity of nicotine detection.

6-3.jpeg

fig.3 the GFP expression in the two detection systems at a nicotine concentration of 0.0001g/L

Model

Sensitivities of the First and the Second Generation Detection Systems

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

By analyzing the experiment data, we get the following models:

6-4.jpeg

We can see that the detected fluorescence of the second generation detection system is higher than the first generation detection system. The difference is most significant at 3h. However, the fluorescence intensity detected by the second-generation system was significantly decreased at 4h. We conjecture that this may be related to the toxic effect of nicotine on the bacteria. Although the first generation of detection system can detect fluorescence, it is not as sensitive as the second generation system.

In conclusion, compared with the first version detector(BBa_K2827005), the second version detector is more sensitive. We should choose the second generation detection system to detect the nicotine concentration and the best detection time is at 3h. However, if the first generation system is selected, the detection is better done after 4 hours, with the optimal detection concentration of less than 0.32 g/L.

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Parameters
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