Difference between revisions of "Team:Tokyo Tech/AHL Assay/AHL Reporter Assay"

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<p class="normal_text"> Our purpose of this experiment is to confirm inducibility of Prhl (BBa_I14017), Plux (BBa_R0062), and Plas(BBa_R0079) promoters upon addition of different AHLs. We sought the best combination of AHL systems for co-culture of Snow White <span style ="font-style : italic"> coli</span> and Queen <span style="font-style : italic">coli</span>. We prepared three plasmids shown below (Fig.3-2-1-5-1) and measured RFU of GFP/Turbidity after adding each AHL.   
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<p class="normal_text"> Our purpose of this experiment is to confirm inducibility of Prhl (BBa_I14017), Plux (BBa_R0062), and Plas (BBa_R0079) promoters upon addition of different AHLs. We sought the best combination of AHL systems for co-culture of Snow White <span style ="font-style : italic"> coli</span> and Queen <span style="font-style : italic">coli</span>. We prepared three plasmids shown below (Fig.3-2-1-5) and measured RFU of GFP/Turbidity after adding each AHL.   
 
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<p class="normal_text"> Our experiments proved that our parts worked as expected (Fig.3-2-1-3-1).<br>
 
<p class="normal_text"> Our experiments proved that our parts worked as expected (Fig.3-2-1-3-1).<br>
 
The ratio of promoter strength was Prhl : Pux : Plas= 2 : 40 :1.<br>
 
The ratio of promoter strength was Prhl : Pux : Plas= 2 : 40 :1.<br>
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The Lux system, which is usually activated by C12, reacted not only to C6 but also C12 (crosstalk). The reaction rate to C12 was half as that of C6. The Rhl system, which is usually activated by C4, reacted not only C4 but also C6 (crosstalk). The reaction rate to C6 was as that of C4.
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The Lux system, which is usually activated by C6, reacted not only to C6 but also C12 (crosstalk). The reaction rate to C12 was half as that of C6. The Rhl system, which is usually activated by C4, reacted not only C4 but also C6 (crosstalk). The reaction rate to C6 was 0.78 times as high as that of C4.
 
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<p class="normal_text"> Considering the expression level, we should use the combination of Lux and Rhl for the final genetic circuits. Furthermore, the results imply that using C12 but not C6 is better to orchestrate the expression controlled by Plux. But it is not enough to endow Prhl the equal promoter strength of Plux. In fact, we simulated the final circuits and found that the Prhl strength was too weak compared to Plux to work the final circuits （see the Modeling page）. Taken together these results, we decided to improve the Prhl to increase its strength (see the Rhl System Assay page).
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<p class="normal_text"> Considering the expression level, we should use the combination of Lux and Rhl for our final genetic circuits. Furthermore, the results imply that using C12 but not C6 is better to orchestrate the expression controlled by Plux. But it is not enough to endow Prhl the equal promoter strength of Plux. In fact, we simulated our final circuits and found that the Prhl strength was too weak compared to Plux to work the final circuits （see the Modeling page）. Taken together these results, we decided to improve the Prhl to increase its strength (see the Rhl System Assay page).
 
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Revision as of 22:35, 13 October 2016

3-2-1 AHL Reporter Assay

1. Introduction

AHLs, which stand for [N-]acyl-homoserine lactones, are small signaling molecules which are employed in “Quorum Sensing” system. In our project, we decided to use three AHLs studied well by several preceding iGEM Teams; the AHL molecules used here were C4HSL (hereafter referred to as C4), 3OC6HSL (C6), and 3OC12HSL (C12).

2. Summary of the Experiment

Our purpose of this experiment is to confirm inducibility of Prhl (BBa_I14017), Plux (BBa_R0062), and Plas (BBa_R0079) promoters upon addition of different AHLs. We sought the best combination of AHL systems for co-culture of Snow White coli and Queen coli. We prepared three plasmids shown below (Fig.3-2-1-5) and measured RFU of GFP/Turbidity after adding each AHL.

3. Results

Our experiments proved that our parts worked as expected (Fig.3-2-1-3-1).
The ratio of promoter strength was Prhl : Pux : Plas= 2 : 40 :1.
The Lux system, which is usually activated by C6, reacted not only to C6 but also C12 (crosstalk). The reaction rate to C12 was half as that of C6. The Rhl system, which is usually activated by C4, reacted not only C4 but also C6 (crosstalk). The reaction rate to C6 was 0.78 times as high as that of C4.


Fig. 3-2-1-3-1 RFU of GFP/ Turbidity of AHL Reporter Assay

4. Discussion

Considering the expression level, we should use the combination of Lux and Rhl for our final genetic circuits. Furthermore, the results imply that using C12 but not C6 is better to orchestrate the expression controlled by Plux. But it is not enough to endow Prhl the equal promoter strength of Plux. In fact, we simulated our final circuits and found that the Prhl strength was too weak compared to Plux to work the final circuits （see the Modeling page）. Taken together these results, we decided to improve the Prhl to increase its strength (see the Rhl System Assay page).

5. Materials and Methods

5-1. Construction

-Strain
All the plasmids were prepared in XL-1 Blue strain.

-Plasmids
A. Pcon-rhlR-Prhl-gfp (pSB6A1) (Fig. 3-2-1-5-1)



Fig. 3-2-1-5-1 Pcon-rhlR-Prhl-gfp (pSB6A1)

B. Pcon-luxR-Plux-gfp (pSB6A1) (Fig. 3-2-1-5-2)



Fig. 3-2-1-5-2 Pcon-rhlR-Prhl-gfp (pSB6A1)

C. Pcon-lasR-Plas-gfp (pBS6A1) (Fig. 3-2-1-5-3)



Fig. 3-2-1-5-3 Pcon-lasR-Plas-gfp (pBS6A1)

D. Pcon-gfp (pSB6A1) …Positive control (Fig. 3-2-1-5-4)



Fig. 3-2-1-5-4 Pcon-gfp (pSB6A1)

E. pSB6A1 …Nagative control (Fig. 3-2-1-5-5)



Fig. 3-2-1-5-5 pSB6A1

-Medium
LB medium A
LB medium containing ampicillin (50 microg/ mL)





5-2. Assay Protocol

The following experiments is performed at 37℃ unless otherwise stated.

1) Prepare overnight cultures for each sample in 3 mL LB medium A with vigorous shaking.

2) Dilute the overnight cultures to 1/60 in fresh LB medium A (1.2 mL).

3) Incubate the fresh cultures for 1 h with vigorous shaking.

4) Add 500 microM C4, 500 microM C6, 500microM C12 or DMSO to each 500 microL sample at the final concentration 10 microM.

5) Incubate the samples for 4 h with vigorous shaking.

6) Add 100 microL of the samples into each well of a plate reader.

7) Measure RFU of GFP/Turbidity at 490 nm as an exciting wavelength, 525 nm as an emission wavelength.

8) Measure the Turbidity at 600 nm.

6. 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