By Levi Clancy for Student Reader on
- Genetic techniques
- 5'-Deletion Mutants
- Ames Test
- Cloning Vectors
- DNA Fingerprinting
- DNA Miniprep
- Gel Shift Assay
- Gene Control in Development: Laboratory Techniques
- Gene Targeting
- Genetic Engineering
- Genetic screen
- In Vitro Nuclear Run-on Experiment
- Interrupted Mating Experiment
- Knockout mutation
- Linkage analysis
- Polymerase Chain Reaction
- Promoter (Transcriptional) (RNA) Fusion
- Reporter Gene
- Restriction Enzymes (Endonucleases )
- Sequence Alignment
- Shotgun sequencing
- Temperature Sensitive Mutant Experiment
- Translational (Protein) Fusion
- Transposon Tagging
- cDNA Microarray
Translational Gene Fusion is used to measure protein localization by quantifying promoter activity.
The fusion results in a gene with a promoter, fluorescent protein gene (usually yellow fluorescent protein YFP or green fluorescent protein GFP), the gene of interest itself and a transcriptional stop codon. There is no stop codon between GFP and the gene of interest, and thus a fusion protein is encoded: our protein of interest is tagged with GFP.
This type of construct allows you to locate the protein of interest within the cell, and detect changes in σ protein stability or activity.
In an experiment where we look at promoter activity we use a GFP to mesaure promoter and protein activity of sigma (σ) factor.
First the GFP gene is introduced onto the σ promoter. Then transcription produces a single mRNA with two fused genes: σ and GFP. Then since there is no stop codon between σ and GFP, translation gives a single polypeptide comprised of 2 fused proteins: σ fused to GFP. The stop codon is placed in the mRNA at the end of the σ gene.
Remember that experimental controls do two things: rule out alternative explanations for results of experiment; and verify that the experimental set-up works and why. A common experimental control is to measure untagged cells for GFP activity, and to cross-check by assaying for mRNA.