Difference between revisions of "Part:BBa K211002:Experience"

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Biosafety concerns

None.

Applications of BBa_K211002

Engineering GPCR signalling pathways.

Diacetyl sensing.

HKUST2009 lab experience of BBa_K211002

HKUST team in 2009 has characterized the BBa_K211002 in the two experiments as shown below. This chimeric odorant sensing GPCR has been proven to localize to the budding yeast membrane and upon binding to diacetyl, triggers downstream signalling pathway.

1.Membrane localization

(1)Protocol 1

Fluorescence microscopy visualization

1. Grow yeast transformed cells containing appropriate plasmids in 5 ml SCM minus His & glucose at 30ºC overnight..

2. Add galactose (to a conc.of 2%) at a cell density corresponding to an OD600 of a 10-fold dilution reaching 0.8.

3. Continue growing cells at 30ºC for 30min to 1.5hr.

4. At time 30min, 60min, 90min, use 1ml of cells, spin down and resuspend cells in 10μl ddH2O.

5. Use 2μl cells and visualize the cells in a fluorescency microscopy.

(2)Result

Upon being induced by galactose for 45mins and 90mins, membrane localization of the chimeric receptor in budding yeast can be clearly observed.

2.Odorant sensing

(1)Protocol 2

FACS

1. Transformed yeast cells are induced with galactose first for 1 hour.

2. Yeasts after 1 hour, at OD600 = 0.8 are induced with diacetyl solution at 5mM.

3. Every 1.5 hour we need to collect samples for FACS analaysis,both control or experiment groups.

4. FACS standard method for sample preparation.

5. Gel pictual for results.

(2)Results

After being induced with galactose, the chimeric receptor is induced with its ligand diacetyl. From 0 to 4 hours, the cells sense the ligand and trigger the downstream mating response, leading to a cell cycle arrest at G1 phase. As a result, the DNA content is shifted towards the G1 non-replicated DNA content. This result is compared with the RI7 receptor (the whole length RI7) induced with hexanal at the same condition.

Fig2. FACS results showing the functional assay of chimeric receptor compared with RI7 receptor. A is the RI7 receptor (1)induced or (2) not induced with the ligand hexanal. B is the chimeric receptor (1)induced or (2) not induced with the ligand diacetyle. 1C and 2C indicates DNA contents.

For more detailed information, please refer to our [http://2009.igem.org/Team:HKUST/OdorantSensing team Wiki].

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