Team:Tokyo Tech/Experiment/Plux and Prhl reporter assay

From 2014.igem.org

Tokyo_Tech

Experiment

Plux and Prhl reporter assay

Contents

1. Introduction

2. Summary of the Experiment

3. Results

4. Materials and Methods

4-1. Construction

4-2. Assay Protocol

5. Reference

 
 

1. Introduction
 

In the modeling (Fig. 3-1-1-1), the better mutualism between Company and Customer requires that the expression of LasI under the control of Prhl promoter (BBa_R0071) is the same level as the expression of RhlI under the control of Plux promoter (BBa_R0062). Thus, we firstly tested the expression level of Prhl promoter and Plux promoter through the reporter assay (Fig. 3-1-1-2).

Fig. 3-1-1-1. The phase diagram of the intensity of Plux and Prhl promoter
Fig. 3-1-1-2. Plux and Prhl reporter assay flow chart
 
 

2. Summary of the Experiment

 

Our purpose is to confirm the expression level of Prhl promoter (BBa_R0071) and Plux promoter (BBa_R0062). We prepared two plasmids sets shown in below (Fig. 3-1-2-1). We measured the fluorescence intensity by GFP expression when we added signaling molecules.

We prepared four conditions shown in below.

A-1: Culture containing Ptet-LuxR and Plux-GFP cell with 3OC12HSL
     A-2: Culture containing Ptet-LuxR and Plux-GFP cell with DMSO
     B-1: Culture containing Ptet-RhlR and Prhl-GFP cell with C4HSL
     B-2: Culture containing Ptet-RhlR and Prhl-GFP cell with DMSO
Fig. 3-1-2-1. Reporter Plasmids
 
 

3. Results
 
Fig. 3-1-3-1. Plux and Prhl Reporter Assay result
 

Fig. 3-1-3-1 shows our experimental data of Plux promoter and Prhl promoter. Compared with the modeling results, the expression level of the native BioBrick Prhl promoter (BBa_R0071) was too weak to satisfy our requirement (Fig. 3-1-3-1 lane 2). In other words, the expression of RhlI under the control of Plux promoter (BBa_R0062) was higher than the expression of LasI under the control of Prhl promoter.

 
 

4. Materials and Methods
 

4-1. Construction

-Strain

All the samples were JM2.300 strain

-Plasmids

A. Ptet-LuxR (pSB6A1), Plux-GFP (pSB3K3)
Fig. 3-1-4-1.

B. Ptet-RhlR (pSB6A1), Prhl-GFP (pSB3K3)

Fig. 3-1-4-2.  
 

4-2. Assay Protocol

  1. 1. Prepare 2 overnight cultures for each sample in 3 mL LB medium, containing ampicillin (50 microg / mL) and kanamycin (30 microg / mL) at 37°C for 12 h.

  2. 2. Dilute the overnight cultures to 1 / 100 in fresh LB medium (3 mL) containing ampicillin (50 microg / mL) and kanamycin (30 microg / mL) (fresh culture).

  3. 3. Incubate the fresh cultures at 37°C until the OD590 reaches 0.3.

  4. 4. Add 30 microL of 500 microM C4HSL, 500 nM 3OC12HSL or DMSO as listed below:

   A-1: A + 500 nM 3OC12HSL
       A-2: A + DMSO
       B-1: B +500 microM C4HSL
       B-2: B + DMSO

  1. 5. Incubate the samples at 37°C for 4 h.

  2. 6. Start preparing the flow cytometer 1 h before the end of incubation.

  3. 7. Take 200 microL of the sample, and centrifuge at 9000x g, 1 min, 4°C.

  4. 8. Remove the supernatant by using P1000 pipette.

  5. 9. Add 1 mL of filtered PBS (phosphate-buffered saline) and suspend.

  6. 10. Dispense all of each suspension into a disposable tube through a cell strainer.

11. Measure the fluorescence intensity with a flow cytometer (We used BD FACSCaliburTM Flow Cytometer of Becton, Dickenson and Company).

 
 

5. Reference

1. 1. Kendall M. Gray et al. (1994) Interchangeability and specificity of components from the quorum-sensing regulatory systems of Vibrio fischeri and Pseudomonas aeruginosa. Journal of Bacteriology 176(10): 3076–3080

 

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