Team:McGill/Experimental

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Experiments
Experiments
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==Basics==
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Two separate cells that secrete transcription factors such that they create a feedback system between each other should interact according to the model detailed in the theory section. That is, oscillatory patterns should become apparent. In order to verify the occurrence of oscillations experimentally, we designed two gene constructs that utilize the Rhl quorum sensing system from P. aeruginosa and the Lux system from V. fischeri.
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===Construct 1===
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*Secretes 3OC6-HSL in <b>the presence</b> of C4-HSL
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*RhlR constitutively expressed.
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[[image:K290003.jpg]]
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[http://partsregistry.org/wiki/index.php?title=Part:BBa_K290003 K290003]
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===Construct 2:===
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*Secretes C4-HSL in <b>the absence</b> of 3OC6-HSL
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*LuxR constitutively expressed.
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[[image:K290002.jpg]]
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[http://partsregistry.org/Part:BBa_K290002 K290002]
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===Plasmid===
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The constructs as synthesized by GenScript were delivered in a copy-number inducible plasmid to minimize mutations. There seems to be instability due to repeats in the STOP sequences which makes complex biobrick constructs hard to synthesize.
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The plasmid in which our constructs were cloned by GenScript is pCC1 from [http://www.epibio.com/main.asp Epicentre]
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===Cells===
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We use the EPI300 cells from [http://www.epibio.com/main.asp Epicentre] that were supplied by GenScript.
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==Planned Experiments==
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===Diffusion of homoserine lactones===
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*Both constructs can be used as receiver devices, one for 3OC6-HSL and the other for C4-HSL. If one has a lawn of cells and pipets a small amount of one of the two molecules in the centre, diffusion rates should be revealed by the rate at which cells turn on (in the case of K290003) or off (for K290002). We plan to determine the rates of diffusion of both molecules, notably for 2% Low-Melt Agarose dissolved in supplemented M9 Medium, as we plan to carry out our experiments in it.
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===Promoter Characterization===
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*In the same line of thought, by having each construct act as a receiver device, we should be able to determine the relationship between the concentration of HSL to the output from each promoter.
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*lux pL driven fluorescence should taper off at higher and higher concentrations of 3OC6-HSL
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*prhl driven fluorescence should increase at higher and higher concentrations of C4-HSL
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*determining the behaviour of each promoter is crucial for better predicting the oscillatory patterns we expect from the full system, which is...
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===Oscillator===
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*The system consists of a simple feedback loop between two (populations of) cells. Theoretical models predict the emergence of oscillatory behaviours in 1 dimension, where the two cells are simply split apart by a certain distance, as well as in 2 dimensions, where cells containing construct 1 are mixed with cells containing construct 2 at varying densities.
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[[image:OscillatorMcGill09.jpg]]

Latest revision as of 03:35, 22 October 2009


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Contents

Experimental Notebook


Experiments

Basics

Two separate cells that secrete transcription factors such that they create a feedback system between each other should interact according to the model detailed in the theory section. That is, oscillatory patterns should become apparent. In order to verify the occurrence of oscillations experimentally, we designed two gene constructs that utilize the Rhl quorum sensing system from P. aeruginosa and the Lux system from V. fischeri.

Construct 1

  • Secretes 3OC6-HSL in the presence of C4-HSL
  • RhlR constitutively expressed.

K290003.jpg

[http://partsregistry.org/wiki/index.php?title=Part:BBa_K290003 K290003]

Construct 2:

  • Secretes C4-HSL in the absence of 3OC6-HSL
  • LuxR constitutively expressed.

K290002.jpg

[http://partsregistry.org/Part:BBa_K290002 K290002]

Plasmid

The constructs as synthesized by GenScript were delivered in a copy-number inducible plasmid to minimize mutations. There seems to be instability due to repeats in the STOP sequences which makes complex biobrick constructs hard to synthesize. The plasmid in which our constructs were cloned by GenScript is pCC1 from [http://www.epibio.com/main.asp Epicentre]


Cells

We use the EPI300 cells from [http://www.epibio.com/main.asp Epicentre] that were supplied by GenScript.

Planned Experiments

Diffusion of homoserine lactones

  • Both constructs can be used as receiver devices, one for 3OC6-HSL and the other for C4-HSL. If one has a lawn of cells and pipets a small amount of one of the two molecules in the centre, diffusion rates should be revealed by the rate at which cells turn on (in the case of K290003) or off (for K290002). We plan to determine the rates of diffusion of both molecules, notably for 2% Low-Melt Agarose dissolved in supplemented M9 Medium, as we plan to carry out our experiments in it.

Promoter Characterization

  • In the same line of thought, by having each construct act as a receiver device, we should be able to determine the relationship between the concentration of HSL to the output from each promoter.
  • lux pL driven fluorescence should taper off at higher and higher concentrations of 3OC6-HSL
  • prhl driven fluorescence should increase at higher and higher concentrations of C4-HSL
  • determining the behaviour of each promoter is crucial for better predicting the oscillatory patterns we expect from the full system, which is...

Oscillator

  • The system consists of a simple feedback loop between two (populations of) cells. Theoretical models predict the emergence of oscillatory behaviours in 1 dimension, where the two cells are simply split apart by a certain distance, as well as in 2 dimensions, where cells containing construct 1 are mixed with cells containing construct 2 at varying densities.

OscillatorMcGill09.jpg

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