Although you’ve used IPTG for your experiments, you may be unfamiliar with the mechanism of IPTG induction. This article provides a quick review on the principle of IPTG induction, how much IPTG to use for induction, and protocols for IPTG induction.
In this article:
What is IPTG?
IPTG or Isopropyl β-D-1-thiogalactopyranoside is a chemical reagent mimicking allolactose, which removes a repressor from the lac operon to induce gene expression.
An allolactose is an isomer of lactose, formed when lactose enters cells. It acts as an inducer to initiate the transcription of genes in the lac operon. The genes in the lac operon encode proteins important for breaking down lactose.
The metabolism of lactose occurs when glucose is absent and lactose is present at high levels. To replenish the energy source, cells naturally break down lactose.
What is the lac operon?
The lac operon is a DNA region of Escherichia coli, containing three genes (lacZ, lacY, and lacA). The three genes, operated under a single promoter, cluster together on the E. coli genome. A promoter is a site where RNA polymerase starts the transcription of a gene.
When lactose is absent, a repressor binds to a lac operator, blocking RNA polymerase and preventing the transcription of the genes. A lac operator is a negative regulatory site on the DNA, overlapping with a lac promoter.
When glucose is absent and lactose is present, allolactose binds to the lac repressor to release it from the lac operator. In a similar way to allolactose, IPTG causes the release of the lac repressor. As a result, RNA polymerase binds to the promoter and gene transcription starts.
What is IPTG Induction?
IPTG induction is a method of regulating protein synthesis by triggering transcription of the lac operon. It requires two key players:
During IPTG induction, cells must produce T7 RNA polymerase required for gene transcription; for example, you can use BL21(DE3) E. coli strain. BL21(DE3) has the lacI gene encoding the lac repressor, LacI. After IPTG induction, this strain expresses T7 RNA polymerase.
In the cloning step, you clone your gene of interest into a particular vector, such as the pET vector, and transform the E. coli cells with the recombinant DNA.
To express your gene of interest, your vector must have several key elements:
- An antibiotic resistance gene: for selecting the transformed cells.
- The lacI gene: for synthesizing the lac repressor, LacI.
- LacO site: a site containing a lac operator sequence. In the absence of IPTG, LacI binds to this site to prevent the expression of your gene of interest.
- T7 promoter (or other promoters recognized by T7 RNA Polymerase): for initiating the transcription of your gene of interest. This promoter is adjacent to the LacO site. Therefore, when LacI binds to the LacO site, it prevents the binding of T7 RNA Polymerase into the T7 promoter.
- ORF (Open Reading Frame): a site where you clone your gene of interest.
How IPTG Induction in E. coli Cells Work
Inside transformed E. coli cells, without the presence of IPTG, LacI binds to the LacO site. The binding of LacI to this site blocks the T7 RNA polymerase from initiating the transcription of your gene of interest. Consequently, there is no production of your protein.
When IPTG is present in the medium, it will enter the cells and remove LacI from the LacO site. As a result, T7 RNA Polymerase binds to the T7 promoter and initiates gene transcription. Eventually, it leads to the synthesis of your protein of interest.
To learn more details about IPTG induction, find the GoldBio article below:
How Much IPTG to Add for Induction
A commonly used protocol would specify how much IPTG to add into growth medium containing the bacterial culture. For GoldBio’s protocols, use 1mM of IPTG in 1 ml of LB medium to make a final concentration of 0.5mM in the medium with bacterial culture. This concentration is sufficient to induce your protein of interest.
How Long Does it Take to Express a Protein for IPTG Induction?
There are two common protocols to induce proteins by IPTG: fast induction and slow induction. For fast induction, you can harvest your protein of interest at least 3-4 hours after IPTG induction. Whereas, for slow induction, you can get your protein at least 12-16 hours post IPTG induction. The reason why you would choose slow induction over fast induction is some proteins are difficult to obtain with fast induction.
Why is IPTG Better than Lactose for Induction?
The bacterial cells can’t process IPTG since it is not the right substrate for the lactose metabolic pathways. Therefore, IPTG remains available in the growth medium for inducing protein expression, instead of being used up as an energy source.
IPTG Induction Protocols
Below are the protocols for fast induction and slow induction:
- Streak out the transformed E. coli strain on a plate and grow the plate overnight at 37°C.Pick a single colony and grow the cells in 15 ml of LB medium containing an antibiotic overnight in a shaking incubator.
- Dilute 1:50 in 2 ml of LB medium with antibiotic and grow 3-4 hours at 37°C in a shaking incubator (Alternatively, dilute 1:100 if you prefer to grow the cells overnight at 37°C).
- Prepare 1 ml LB with an antibiotic and 1mM of IPTG in a 15 ml conical tubes and prewarm it for 10 minutes at 37°C before use.
- After 3-4 hours, remove 1 ml from the bacterial culture and place in a labeled 1.5 ml tube. Spin at maximum speed for 30 seconds at room temperature, remove supernatant, and freeze at -20°C until needed. This is the uninduced control.
- Add prewarmed 1 ml LB+antibiotic+1mM IPTG into the tube containing the bacterial culture and grow to 37°C for 3-4 hours. In this tube, the total volume is 2 ml and the final concentration of IPTG is 0.5mM.
- After 3-4 hours post IPTG induction, transfer 1 ml to labeled 1.5 ml tubes and spin at maximum speed at room temperature for 30 seconds. Remove the supernatant and freeze the pellet at -20°C until needed. This is the induced control.
- Prepare sample for SDS page.
- Follow step 1-4 from the fast induction protocol.
- Add 1 ml LB+antibiotic+1mM IPTG (prewarmed to 20°C) into the tube containing the bacterial culture and grow at 20°C for 12 to 16 hours. In this tube, the total volume is 2 ml and the final concentration of IPTG is 0.5mM.
- After 12-16 hours post IPTG induction, transfer 1 ml from induced sample to labeled 1.5 ml tubes and spin at maximum speed for 30 seconds at room temperature. Remove the supernatant and freeze the pellet at -20°C until needed. This is the induced control.
- Prepare sample for SDS Page.
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