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Team:McGill/Experimental
From 2009.igem.org
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 response to C4-HSL
- RhlR constitutively expressed.
[http://partsregistry.org/wiki/index.php?title=Part:BBa_K290003 K290003]
Construct 2:
- Secretes C4-HSL in response to COC6-HSL
- LuxR constitutively expressed.
[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, especially for 2% Low-Melt Agarose dissolved in supplemented minimal medium, as we plan to carry out our experiments in such a medium.
