Team:Tokyo Tech/Project/Artificial Cooperation System/lux act rep/Pluxrep/assay1

From 2010.igem.org

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We constructed K395101 combining R0061 and K121013. K121013 is a promoter-less gfp reporter (rbs-gfp-ter-ter) on pSB6A1. S03119 is a LuxR generator which is regulated by PtetR, which is repressed by TetR. In this experiment, we don’t use TetR, so, S03119 functions a LuxR constitutive generator. The backbone of S03119 is pSB1A2, which is a high copy plasmid, so we changed the backbone from pSB1A2 to pSB3K3.
We constructed K395101 combining R0061 and K121013. K121013 is a promoter-less gfp reporter (rbs-gfp-ter-ter) on pSB6A1. S03119 is a LuxR generator which is regulated by PtetR, which is repressed by TetR. In this experiment, we don’t use TetR, so, S03119 functions a LuxR constitutive generator. The backbone of S03119 is pSB1A2, which is a high copy plasmid, so we changed the backbone from pSB1A2 to pSB3K3.
We used a fusion of PlacI<sup>q</sup> (I14032) to gfp (K121013) as a positive control and used promoterless gfp (K121013) as a negative control.
We used a fusion of PlacI<sup>q</sup> (I14032) to gfp (K121013) as a positive control and used promoterless gfp (K121013) as a negative control.
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'''samples'''
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*'''samples'''
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1-1. [Plux rep - GFP](BBa_K395101) on pSB6A1 + [PtetR – LuxR] on pSB3K3
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#[Plux rep - GFP](BBa_K395101) on pSB6A1 + [PtetR – LuxR] on pSB3K3
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1-2. positive control: [PlacI<sup>q</sup>(constitutive promoter) - GFP] on pSB6A1+ [PtetR – LuxR] on pSB3K3  
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#positive control: [PlacI<sup>q</sup>(constitutive promoter) - GFP] on pSB6A1+ [PtetR – LuxR] on pSB3K3  
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1-3. negative control: [promoterless - GFP] on pSB6A1+ [PtetR – LuxR] on pSB3K3
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#negative control: [promoterless - GFP] on pSB6A1+ [PtetR – LuxR] on pSB3K3
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'''Strain'''
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*'''Strain'''
DH5&alpha
DH5&alpha
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'''protocol'''
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*'''protocol'''
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1. Prepare overnight culture.
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#Prepare overnight culture.
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2. Take 30 ul of the overnight culture into LB + antibiotics (Amp + Kan).
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#Take 30 ul of the overnight culture into LB + antibiotics (Amp + Kan).
(→fresh culture)
(→fresh culture)
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3. Incubate the fresh culture until the observed O.D. reaches around 0.80.
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#Incubate the fresh culture until the observed O.D. reaches around 0.80.
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4. Each sample was divided into 2.
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#Each sample was divided into 2.
Prepare and add 3OC6HSL mixture to one, and add DMSO mixture to the other.
Prepare and add 3OC6HSL mixture to one, and add DMSO mixture to the other.
The final concentration of AHL is 100nM.
The final concentration of AHL is 100nM.
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5. Induction for 2 hours at 37°C.
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#Induction for 2 hours at 37°C.
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6. Fluorometer (FLA5200) and flow cytometry measurements for GFP expression.
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#Fluorometer (FLA5200) and flow cytometry measurements for GFP expression.
===Reference===
===Reference===

Revision as of 13:50, 26 October 2010

iGEM Tokyo Tech 2010 "E.coli with Humanity"

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Contents

luxR repression promoter (BBa_R0061) assay

Abstract

We characterized the strength of this promoter which has never been done before in BioBrick in order to design new promoter based on this data. We measured fluorescence by flow cytometry 3 hour after addition of 100nM 3OC6HSL to confirm R0061, which is an existing BioBrick promoter repressed by LuxR/3OC6HSL complex.

Introduction

We characterized the strength of this promoter which has never been done before in BioBrick in order to design new promoter base on this data. In the Artificial Cooperation System, we inserted chloramphenicol resistance coding sequence to this promoter. Thus, this promoter plays an important role in Artificial Cooperation System.

Results

The result is shown in fig.○. The expression of GFP with 3OC6HSL dropped to 1/3 comparing with the expression without 3OC6HSL.

Conclusion

We confirmed that 3OC6HSL repressed luxR repression promoter, R0061, as expected.

Materials & Methods

We constructed K395101 combining R0061 and K121013. K121013 is a promoter-less gfp reporter (rbs-gfp-ter-ter) on pSB6A1. S03119 is a LuxR generator which is regulated by PtetR, which is repressed by TetR. In this experiment, we don’t use TetR, so, S03119 functions a LuxR constitutive generator. The backbone of S03119 is pSB1A2, which is a high copy plasmid, so we changed the backbone from pSB1A2 to pSB3K3. We used a fusion of PlacIq (I14032) to gfp (K121013) as a positive control and used promoterless gfp (K121013) as a negative control.

  • samples
  1. [Plux rep - GFP](BBa_K395101) on pSB6A1 + [PtetR – LuxR] on pSB3K3
  2. positive control: [PlacIq(constitutive promoter) - GFP] on pSB6A1+ [PtetR – LuxR] on pSB3K3
  3. negative control: [promoterless - GFP] on pSB6A1+ [PtetR – LuxR] on pSB3K3
  • Strain

DH5&alpha

  • protocol
  1. Prepare overnight culture.
  2. Take 30 ul of the overnight culture into LB + antibiotics (Amp + Kan).

(→fresh culture)

  1. Incubate the fresh culture until the observed O.D. reaches around 0.80.
  2. Each sample was divided into 2.

Prepare and add 3OC6HSL mixture to one, and add DMSO mixture to the other. The final concentration of AHL is 100nM.

  1. Induction for 2 hours at 37°C.
  2. Fluorometer (FLA5200) and flow cytometry measurements for GFP expression.

Reference

1. KRISTI A. EGLAND & E. P. GREENBERG Conversion of the Vibrio fischeri Transcriptional Activator, LuxR, to a Repressor JOURNAL OF BACTERIOLOGY, Feb. 2000, p. 805–811



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