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

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                     <p class="info-24"><a href="#Introduction">1. Introduction</a></p>
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                     <p class="info-24"><a href="#Assay">4-2 Assay Protocol</a></p>
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Revision as of 10:06, 17 October 2014

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 interdependence 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 by the reporter assay (Fig. 3-1-1-2.).

Fig. 3-1-1-1. The phase diagram of Plux and Prhl intensity
Fig. 3-1-1-2. Plux and Prhl Reporter Assay flow chart
 

2. Summary of the experiment

 

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

We prepared four conditions as follow.

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
 

The measured activity of pre-existing Biobrick Prhl promoter (BBa_R0071) was too weak to satisfy the requirement from our modeling results. (Fig. 3-1-3-1 lane 2) In other words, the expression of RhlI under the control of Plux promoter (BBa_R0062) is 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 of every sample A~D 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). Make glycerol stocks from the remainders.

  3. 3. Incubate the fresh cultures in 37°C until the observed OD590 reaches 0.3 (Actual value 0.42).

  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 9000 Xg, 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 fluorescence intensity with a flow cytometer (We used BD FACSCaliburTM Flow Cytometer of Becton, Dickenson and Company).

5. Reference

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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