Why are inducible expression systems used?
Inducible expression sytsems has been developed in many organism (yeast, plants, bacteria, and animals)
- Required in basic research and biotechnology
- to study the function of genes or to produces as much as possible of a prduct
- allow activating a gene of interest at a defined time point, so that the effect of that gene would only manifest after its induction
Note: constitutive expression of a gene can have negative effects, for example it may limit cell proliferation or trigger secondary effects not intended
Principle of the estradiol (EST)-inducible system
EST is a 2 component system
The two genes are normally located close on the chromosome of the transgenic organism
- LexA: DNA binding domain of bacterial repressor LexA from E.coli
- VP16: a strong transcriptional activator domain from the herpex simlplex virus
- hER: C-terminal region of human estrogen receptor
- XVE: a chimeric TF
The TF XVE is constitutively expressed and contains of a
- LexA DNA binding domain
- a VP16 transcriptional activator
- a C-Terminal region from the human estrogen receptor.
Only when estradiol is introduced it binds estradiol and is activated.
The activated form of XVE is then able to bind to the promotor region of the Gene of Interest (e.g.: another TF) whose expression is then induced.
The expressed GOI can then act in the cell and the biological effects can be observed.
–> This method can be used for the inducible expression of any gene, including those encoding TFs
use of synthetic nuclear hormone receptors for inducible gene expression
Nuclear Hormone Receptors: Is a TF located in the Cytoplasm bound by an HSP90.
Only after the Ligand-binding (e.g Estradiol) the HSP90 is removed and the nuclear hormone receptor can enter the nucleus and regulate the GOI expression.
What is the difference between a two-component and a one-component system?
One-component system:
* only onegene must be inserted into the genome of the organism
* Can only be used for nuclear proteins, like TFs
Two-component system:
* both elemts must be interted into the genome of the organism
* can be used for inducible expression of any gene, including those encoding TFs
How does the dexamethasone (DEX)-inducible system work?
One-Component system
- A TF-GR fusion protein is constitutively and strongly expressed.
- The Protein is located in the cytoplasm in the absence of DEX, the TF cannot regulate its target genes in the nucleus.
- Only after The addition of the ligand (DEX) the TF-GR fusion protein translocates into the nucleus.
- The TF can now regulate its Target Genes –> biological effects can be observed.
How does the ethanol-inducible system work?
Two-Component-system
–> both elemnts must be insterted into the genome of the organism
- The TF AlcR is constitutively expressed
- its a zinc-finger-type TF that requires ethanol for activation.
- Only when ethanol is introduced it binds the ethanol and is activated.
- The activated form of AlcR is then able to bind to the promotor region of the Gene of Interest (e.g.: another TF) whose expression is then induced.
- The expressed GOI can then act in the cell and the biological effects can be observed.
Potential Problem: Ethanol itself is a metabolite in many organisms; thus, if ethanol is produced in the organism studied, it might affect the data obtained. This must be kept in mind when using ethanol as an inducer of gene expression
How do the Tet-based repression and activation systems work?
Basic Element:
* Tet repressor from E.coli (Tn10), which is part of the tetracycline resistance operon
Tet-based system is used in two versions:
1. As a promoter-repressing system
The TET-repressor from E.Coli binds to the operator and prevents transcription.
When TET is introduced, it bind the repressor and the repressor dissociates from the operator allowing for native expression.
Problem: Continuous presence of TET is needed for activation, high levels of TetR are needed for sufficient repression in absence of
2. As a promoter-activating system
TetR is fused to VP16 (activation domain) –> tTA (tetracyclin transactivator).
When tTA binds to the operator = transcription is induced.
When TET is present it binds to tTA leading to the disassociation of the complex from the operator = Transcription is stopped.
Problem: native expression can take place.
What is Optogenetics?
light-inducible gene expression
–> Light can be easily switched on and off, allowing the activation or stop of a transcriptional activity
What are chemical inducers?
- EST, Ethanol, Tet
but they have a downside:
Once they are added to the medium (in which cells grow) or to the animal, it is
difficult/time consuming to remove them again to initiate the inactivation of the gene
What is the principle of the red light-controlled gene expression system?
- Light-sensitive proteins interact with TF
- Interaction is reversible depending on light-wavelength.
In Plants:
* Phytochromes interact with PIF (TF)
* Red light: 660 nm: PhyB and PIF form PhyB-PIF complex (active) that can bind promotor of target genes.
* Phy-Proteins need a chromophore to be functional (PCB).
* PCB needs to be added to medium or be present in organism.
* PhyB has the DNA-binding-Domain.
* PIF has the Activation Domain.
* Darkness or >720nm reverse this reaction
