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luxR activation promoter (BBa_R0062) assay2

In a concentration gradient of 3OC6HSL

Abstract

After measurement of the fluorescence intensity of R0062 in case of presence/absence of 3OC6HSL, we measured the 3OC6HSL concentration dependence of Plux activity. We measured the fluorescence intensity under different concentration of 3OC6HSL (0nM, 1nM, 3nM, 5nM, 10nM, 30nM, 50nM, 100nM by flow cytometry 3 hours after 3OC6HSL induction.

Introduction

We confirmed R0062 is activated by LuxR/3OC6HSL complex. As we mentioned before, in Artificial Cooperation System, threshold of promoters regulated by AHL is greatly important. Therefore, we confirmed the how the luxR activation promoter activity depends on the concentration of 3OC6HSL.

Results

The result is shown in fig.○. This result indicates that fluorescence intensity of luxR activation promoter is dependent on 3OC6HSL concentration.

Conclusion

The threshold of fluorescence intensity of R0062, luxR activation promoter regulated by 3OC6HSL is around 5nM.

Materials & Methods

• samples

1. [Plux act - GFP](BBa_K395100) on pSB6A1 + [ptet – LuxR] on pSB3K3

• Strain

DH5&alpha

• protocol

1. Prepare overnight culture.
2. Take 30 ul of the overnight culture into LB + antibiotics (Amp + Kan). (→fresh culture) Prepare the same 7 tubes for each sample.
3. Incubate the fresh culture until the observed O.D. reaches around 0.60.
4. Each sample was divided into 2. Prepare and add 3OC6HSL mixture. The final concentration of 3OC6HSL is 1, 3, 5, 10, 30, 50, 100nM.
5. Induction for 3 hours at 37°C.
6. Fluorometer (FLA5200) and flow cytometry measurements for GFP expression.