Team:Tokyo Tech/Project/Artificial Cooperation System/lux act rep/Pluxrep/assay2

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=luxR repression promoter (BBa_K395008) assay=
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===Abstract===
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We wanted to characterize the strength of this promoter which we designed. We measured fluorescence by flow cytometry 3 hour after addition of 100nM 3OC6HSL to confirm K395008, which is a promoter repressed by LuxR/3OC6HSL complex.
 +
===Introduction===
 +
Even subtle changes in promoter may have distinct effects on the expression of gene. We designed a new promoter, K395008, which is repressed by LuxR/3OC6HSL complex by changing one base of the existing BioBrick parts (BBa_R0061). We wanted to characterize this luxR repression promoter. Also, we wanted to confirm that this promoter is also repressed by LuxR/3OC6HSL complex but has different strength from the existing BioBrick part.
 +
===Results===
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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, K395008, as expected.
 +
===Materials & Methods===
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We constructed K395105 combining K395008 and K121013. K121013 is a promoter-less gfp reporter (rbs-gfp-ter-ter) and this backbone is pSB6A1. Promoter of S03119 is 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.
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[[IMAGE:Tokyotech_K395008assay_construction.png‎|400px]]
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*samples
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#[R0061weak - GFP](BBa_K395105) 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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#negative control: [promoterless - GFP] on pSB6A1+ [PtetR – LuxR] on pSB3K3
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*Strain
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DH5&alpha
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*protocol
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#Prepare overnight culture.
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#Take 30 ul of the overnight culture into LB + antibiotics (Amp + Kan). (→fresh culture)
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#Incubate the fresh culture until the observed O.D. reaches around 0.60.
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#Each sample was divided into 2. Prepare and add 3OC6HSL mixture to one, and add DMSO mixture to the other. The final concentration of 3OC6HSL is 100nM.
 +
#Induction for 3 hours at 37°C.
 +
#Fluorometer (FLA5200) and flow cytometry measurements for GFP expression.
 +
 +
===Reference===
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#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

Latest revision as of 14:39, 26 October 2010

iGEM Tokyo Tech 2010 "E.coli with Humanity"

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Contents

luxR repression promoter (BBa_K395008) assay

Abstract

We wanted to characterize the strength of this promoter which we designed. We measured fluorescence by flow cytometry 3 hour after addition of 100nM 3OC6HSL to confirm K395008, which is a promoter repressed by LuxR/3OC6HSL complex.

Introduction

Even subtle changes in promoter may have distinct effects on the expression of gene. We designed a new promoter, K395008, which is repressed by LuxR/3OC6HSL complex by changing one base of the existing BioBrick parts (BBa_R0061). We wanted to characterize this luxR repression promoter. Also, we wanted to confirm that this promoter is also repressed by LuxR/3OC6HSL complex but has different strength from the existing BioBrick part.

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, K395008, as expected.

Materials & Methods

We constructed K395105 combining K395008 and K121013. K121013 is a promoter-less gfp reporter (rbs-gfp-ter-ter) and this backbone is pSB6A1. Promoter of S03119 is 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.

Tokyotech K395008assay construction.png

  • samples
  1. [R0061weak - GFP](BBa_K395105) 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)
  3. Incubate the fresh culture until the observed O.D. reaches around 0.60.
  4. Each sample was divided into 2. Prepare and add 3OC6HSL mixture to one, and add DMSO mixture to the other. The final concentration of 3OC6HSL is 100nM.
  5. Induction for 3 hours at 37°C.
  6. 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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