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

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3) Add C4 (100 microM) into Queen-like <span style="font-style : italic">coli</span> and C12 into Snow White-like <span style="font-style : italic">coli</span>, and incubate them at 700 rpm for 4 h.<br><br>
 
3) Add C4 (100 microM) into Queen-like <span style="font-style : italic">coli</span> and C12 into Snow White-like <span style="font-style : italic">coli</span>, and incubate them at 700 rpm for 4 h.<br><br>
  
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4) Measure RFU of GFP / Turbidity of GFP at 490nm as an exciting wavelength, 525nm as a measurement wavelength.  
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4) Measure the fluorescence intensity of GFP at 490nm as an exciting wavelength, 525nm as a measurement wavelength.  
  
 
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Revision as of 05:56, 14 October 2016

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

1. Introduction

Our final genetic circuits include TA and Quorum Sensing systems (Fig.3-2-2-1) as well as other inducible promoters and genes. The whole circuits were complicated, and thus, we divided the circuits into some modules and evaluated functionality of each module. In this section, the module of Quorum Sensing is evaluated; production of AHLs and communication between Snow White coli and Queen coli though AHLs was observed.



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 normal Prhl.

3. Results

RFU of GFP / Turbidity was almost same in spite of adding C4 or 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 below, we again recognized that we have to improve Prhl strength to operate our final circuits as we expect.

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