Biology – Principles of genetic technology | e-Consult

To confirm gene expression in yeast using a GFP fusion, the researcher would follow these steps:

1. Transformation: Transform competent yeast cells with the plasmid containing the gene of interest fused to GFP. This involves introducing the plasmid into the yeast cells, allowing them to take it up.
2. Selection: Select for transformed cells using a selective media (e.g., media containing an antibiotic resistance gene present on the plasmid). This ensures that only cells that have taken up the plasmid survive and grow.
3. Microscopy: Examine the transformed yeast cells under a fluorescence microscope. Excited with the appropriate wavelength of light, the GFP will emit green fluorescence.
4. Controls: Crucially, the researcher must include appropriate controls:

   • Negative Control (No GFP): Transform yeast cells with an empty vector (a vector without the GFP fusion). This helps to identify any background fluorescence or non-specific binding.
   • Positive Control (Known Expressing Gene): Transform yeast cells with a plasmid containing a gene known to be expressed under the same conditions (e.g., a reporter gene under the control of a known promoter). This confirms that the experimental setup is working correctly.

5. Interpretation:

   • If the transformed yeast cells show green fluorescence under the fluorescence microscope, it indicates that the gene of interest is being expressed.
   • The intensity of the fluorescence can be used to estimate the level of gene expression.
   • Comparing the fluorescence intensity of the transformed cells with the negative control (no GFP) and the positive control (known expressing gene) allows the researcher to determine whether the GFP fusion is specifically reflecting the expression of the gene of interest. A strong signal in the transformed cells but not in the negative control suggests successful gene expression.