Tetracycline-controlled gene expression relies on a tightly regulated system involving a Tet repressor protein (TetR) and a Tet operator sequence (tetO). TetR naturally binds to tetO, preventing transcription. However, the addition of tetracycline (or its analog doxycycline) alters TetR’s conformation, preventing its binding to tetO.
This absence of TetR allows RNA polymerase to access the promoter, initiating transcription of the downstream gene. Conversely, removing tetracycline allows TetR to rebind tetO, effectively silencing gene expression. The system’s sensitivity depends on the specific TetR variant and promoter design. Stronger promoters result in greater gene expression upon induction.
Several variations exist, including the reverse Tet system where tetracycline *inhibits* transcription. This involves a modified TetR variant that only binds tetO in the presence of tetracycline. Choosing the appropriate system depends on the desired outcome; activation or repression of target gene expression.
Careful consideration should be given to TetR concentration and tetracycline dosage. High TetR levels can lead to leakiness (low-level expression even without induction), while insufficient TetR results in ineffective repression. Optimizing these parameters is crucial for achieving tight control.
Furthermore, the system’s performance can be affected by cell type and gene copy number. Higher gene copy numbers generally lead to increased expression levels, but may also increase background “leakiness”. This highlights the need for optimization within the specific experimental context.
