Team:Tokyo Tech/AHL Assay/Only Assay

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3-2-2 AHL Only of Final genetic circuits Assay

Contents

1. Introduction

2. Summary of the Experiment

3. Results

4. Discussion

5. Materials and Methods

   5-1. Construction

   5-2. Assay Protocol

6. Reference

1. Introduction

　This section is continued from the previous section (3-2-1 AHL Reporter Assay), and the point of Quorum Sensing was further evaluated. Specifically, production of AHLs and communication between Snow White coli and Queen coli though AHLs was analyzed.

Fig. 3-2-2-1 Our final genetic circuits

2. Summary of the Experiment

The reporter (Queen-like? nnn) and sender (Snow White-like? nnn) E. coli cells were prepared which carried nnn and nnn genes, respectively. Then,(a) C4 was added into Queen-like coli, and (b) C4 was added into co-culture of Queen-like coli and Snow White-like coli. The RFUs of GFP and RFP were measured, indicating that Prhl activity was so low that our final genetic circuits would not work with wild type Prhl.

3. Results

RFU of GFP / Turbidity was almost same in spite of adding C4 or DMSO (negative control; note that C4 was dissolved with DMSO) into Queen-like coli or co-culture (Fig.3-2-2-3). The leak of Prhl was high and its cause noticed at Rhl System Assay page.

Fig. 3-2-2-2 RFU of GFP / Turbidity of Only Assay

4. Discussion

The expression level of Prhl was so weak and the leak was so high that Queen-like coli could not influence to Snow White-like coli; in other words, our final genetic circuits did not work. Taken together the results, we again recognized that we have to improve Prhl strength to operate our final circuits.

5. Materials and Methods

5-1. Construction

-Strain All the plasmids were prepared in XL1-Blue strain.
-Plasmids A. Pcon-rbs-rhlR-LVA(pSB6A1), Prhl-rbs-lasI-rbs-gfp-LVA (pSB3K3) (Fig. 3-2-2-5-1)

Fig. 3-2-2-5-1 Pcon-rbs-rhlR-LVA(pSB6A1), Prhl-rbs-lasI-rbs-gfp-ssrA (pSB3K3)

B. Pcon-rbs-luxI(pSB6A1), Plux-rbs-rhlI-rbs-rfp-ssrA (pSB3K3) (Fig. 3-2-2-5-2)

Fig. 3-2-2-5-2 Pcon-rbs-luxI(pSB6A1), Plux-rbs-rhlI-rbs-rfp-ssrA (pSB3K3)

C. pSB6A1, pSB3K3…Negative (Fig. 3-2-2-5-3)

Fig. 3-2-2-5-3 pSB6A1, pSB3K3

-Medium
LB medium AK
LB medium containing ampicillin (50 microg/ mL) and kanamycin (50 microg/ mL)

5-2. Assay Protocol

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

1) Prepare the overnight cultures in LB medium AK at 180 rpm.

2) Dilute the overnight cultures to 1/60 in fresh LB medium AK at 700 rpm for 1 h.

3) Add C4 (100 microM) into Queen-like coli and C12 into Snow White-like coli, and incubate them at 700 rpm for 4 h.

4) Measure the fluorescence intensity of GFP at 490nm as an exciting wavelength, 525nm as a measurement wavelength.

6. Reference

(1) Bo Hu et al. (2010) An Environment-Sensitive Synthetic Microbial Ecosystem. PLoS ONE 5(5): e10619
(2) Chen M. Zhang et al (2014) Distributed implementation of the genetic double-branch structure in Escherichia coli. Chinese Science Bulletin 59: 4625-4630