PDF Version of a Deep Dive Into IPTG Induction

IPTG induction is a longstanding technique in molecular biology. In this article, you’ll take a deep dive into this important application. You will learn what IPTG is, what induction is, who the main characters are, IPTG’s role in induction and the steps that take place, along with a lot more. If you’re new to the topic or just want a refresher, buckle up…






What is IPTG (Isopropyl-beta-D-thiogalactoside)?

Since you’re learning or getting a refresher on IPTG induction, let’s start at the most important place and understand what IPTG is.

IPTG is the structural analog of lactose. While lactose is part of the E. coli's metabolic pathway, IPTG is not. It is this nonmetabolic property that makes it an ideal reagent with working with E. coli in the lab because IPTG won't be broken down.

Within a laboratory setting, the most primary uses for IPTG are in blue/white colony screening and for the induction of recombinant proteins (the focus of this article).

lactose molecular structure vs. IPTG molecular structure




What is Protein Induction?

Great, now you know what IPTG is and that it’s used for protein induction – but what is protein induction? Think of the word induce on its own. The word means to “lead or move by persuasion or influence, as to some action or state of mind.” Therefore, in the scenario of IPTG, its role is to influence or induce protein expression.




Who are the Main Characters in IPTG Induction?

If you’ve done some research on IPTG and induction, you’re going to have had some words thrown at you like lac operon, lac repressor, lactose, etc. Rather than flood you with these words throughout the article, it might be helpful to look at each thing and its role individually. Then once you delve deeper and learn about the process and steps of IPTG induction, you’ll have an easy, mental “who’s who” to keep everything in frame.

The major players of protein induction with IPTG and their role:

  • IPTG – structurally mimics lactose and is used to induce protein expression.
  • DE3 E. coli Strain – A commonly used E. coli strain for protein expression.
  • E. coli RNA Polymerase – E. coli's own RNA polymerase that is used for the strain to copy DNA into RNA in the transcription process.
  • pET Expression Vector – The pET expression vector is an engineered plasmid that uses the origin of replication from pBR322 and the basis for the T7 phage RNA polymerase promoter for recombinant protein expression on the lac operon.
  • T7 RNA Polymerase – T7 RNA polymerase is highly specific for its own promoter, solely transcribing DNA located downstream of the T7 promoter.
  • Lac Repressor- The Lac repressor (LacI) inhibits genes that code proteins involved in bacterial lactose metabolism. In the case of IPTG induction, when IPTG is not present, the lac repressor will prevent E. coli’s RNA polymerase from transcribing T7 RNA polymerase.
  • Lac Operon – A typical operon is a cluster of genes that remains under the control of a single promoter. It usually is made up of a promoter, operator and structural genes. The lac operon in E. coli, therefore, is required for and regulated by the lactose metabolism (which IPTG would substitute). It’s really important to note that the lac operon is found on both the DE3 E. coli genome as well as the pET vector.
  • Gene of Interest – The gene you want expressed. This is the gene of study that is inserted into your vector prior to the vector being taken up by E. coli (in this article the vector would be the pET expression vector).



What are the Steps Involved in Induction? (This is what you’ve been waiting for)

Now that we’re clear on what’s what and who’s who, let’s look at exactly what’s going on in induction: what’s happening, how it’s happening and when it’s happening…

Preparation

The first part of protein induction is just making sure you have all your ducks in a row. That means having your vector prepared and ready, making sure your cells are competent and then getting your cells to take up the vector. Let’s look at that in a more stepwise fashion though:

  • You’ll start protein induction with a commercially available vector, usually pET where your gene of interest would be inserted. This very small vector will have coding for several things, including your antibiotic resistance gene, the ever-important lac repressor gene from the lac operon and of course the gene you want expressed.
  • Next, if you’re not working with competent E. coli cells, which many commercially available DE3 strains are already competent, you’ll have to prepare your cells.
  • Finally, you need your E. coli to take up that vector, in this case, the DE3 E. coli strain. This would happen either by chemical transformation or electroporation.

Inside E. coli

Inside the E.coli, which now contains your vector, the real action of protein induction starts to happen. Your E. coli that took up the vector will eventually divide, and in doing so the daughter cells will have its parent genome as well as copies of the pET vector. Below is what starts to happen in these cells and why IPTG is so important:

  • 1On the lac operon within the E. coli genome (remember the lac O is found on both DE3 and pET), there is a binding site for E. coli’s RNA polymerase. However, since the lac operon relies on lactose or IPTG for anything to happen, in its absence, the lac repressor will bind to that site instead, preventing RNA polymerase from working. When IPTG is present (no one would really use lactose for induction), an important conformational change occurs, causing the lac repressor to fall off and for E. coli’s RNA polymerase to start transcribing the T7 gene for T7 RNA polymerase.
  • Now we move to the lac operon found on the pET vector where transcription at that promoter will only occur by the hand of T7 RNA polymerase. However, since a lac operon is present, once again, when IPTG is not present, the lac repressor is going to bind to the binding site, preventing transcription. Once IPTG is present, it will cause a conformational change and the lac repressor dissociate. The T7 RNA polymerase, which is going to bind to the binding site, and expression of your target gene can finally happen.

If you are interested in learning about other aspects of protein expression, make sure to check out this troubleshooting article, and take a look at some of our high-quality products for expression and purification, including IPTG.



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References:

Arur, S., & Nayak, S. (n.d.). IPTG Induction. Retrieved August 19, 2016.

Beel, C. E., & Lewis, M. (2000, March 7). A closer view of the conformation of the Lac repressor bound to operator. Nature Structural & Molecular Biology, 209-214. doi:10.1038/73317

Gay, G., Wagner, D. T., Keating-Clay, A. T., & Gay, D. C. (2014, October 7). Rapid modification of the pET-28 expression vector for ligation independent cloning using homologous recombination in Saccharomyces cerevisiae. Plasmid. doi:10.106/j.plasmid.2014.09.005

Lewis, M., Chang, G., Horton, N. C., Kercher, M. A., Pace, H. C., Schumacher, M. A., . . . Lu, P. (1996). Crystal Structure of the Lactose Operon Repressor and Its Complexes with DNA and Inducer. Science, 271(5253), 1247-1254. doi:10.1126/science.271.5253.1247

Otten, M., Ott, W., Jobst, M. A., Milles, L. F., Verdorfer, T., Pippig, D. A., . . . Gaub, H. E. (2014, September 07). From genes to protein mechanics on a chip. Nature - Methods, 1127-1130. doi:10.1038/nmeth.3099

Ramos, C., Abreu, P., Nascimento, A., & Ho, P. (2004). A high-copy T7 Escherichia coli expression vector for the production of recombinant proteins with a minimal N-terminal His-tagged fusion peptide. Braz J Med Biol Res Brazilian Journal of Medical and Biological Research, 37(8). doi:10.1590/s0100-879x2004000800001

Siegel, A. (2011, September 16). How does IPTG induced gene expression work at a molecular level? Retrieved August 16, 2016.

Tabor, S., & Richardson, C. C. (1985). A bacteriophage T7 RNA polymerase/promoter system for controlled exclusive expression of specific genes. Proceedings of the National Academy of Sciences, 82(4), 1074-1078. doi:10.1073/pnas.82.4.1074


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