Team:Tokyo Tech/AHL 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

Our final genetic circuits are combined TA system and Quorum Sensing (Fig.3-2-3-1). Constructing complicated circuits needs assays divided into each part. This assay is about a part of Quorum Sensing and focused on production and reception of AHL to express the communication between Snow White coli and Queen coli.

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

2. Summary of the Experiment

Our purpose is to confirm the circuits working as a reporter and a sender to specific AHL. Add (a) C4 into Queen-like coli, (b) C4 into co-culture of Queen-like coli and Sow White-like coli. Then measure the RFU of GFP and RFU of RFP. The results indicate that Prhl strength is so low that our final genetic circuits cannot work with normal Prhl.

3. Results

RFU of GFP/ Turbidity was almost same in spite of adding C4 or DMSO into Queen-like 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 high that Queen-like coli could not influence to Snow White-like coli, in other words, our final genetic circuits could not work. Taken together the results below, we have to improve Prhl strength to work our final circuits.

5. Materials and Methods

5-1. Construction

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

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

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

Fig. 3-2-2-5-2 Pcon-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