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…




No time to read the whole thing? Look to this list as a reference for topics covered and scroll to the piece of information you need most.

  • What is IPTG
  • What is Protein Induction
  • Who are the “Main Characters” (a who/what section for induction)
  • What are the Steps Involved in Induction (the most vital information)
  • Why You Can’t Use Lactose for Induction
  • General Optimization Information



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 – the why it’s important will naturally come later in the article.

IPTG is the structural analog of lactose; however within a cell, it is not part E. coli’s metabolic pathway. Its nonmetabolic property makes it ideal in the lab since it 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).



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.

A closer look at IPTG Induction. Protein Induction.




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 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 PolymeraseE. coli’s own RNA polymerase that will be used for the E. coli genome.
  • 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 – a highly selective RNA polymerase that selects for its own promoter. In protein induction with IPTG, T7 RNA polymerase transcription and translation is what requires initial IPTG induction.
  • 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 (lactose structural analog), the lac repressor will prevent E. coli’s RNA polymerase from transcribing T7 RNA polymerase.
  • Lac Operon – Recall that 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 – This is what you want expressed when all is said and done, and the gene itself is inserted into your vector prior to the vector being taken up by E. coli (in this article that would be the pET expression vector).

Use the illustration at the top of the page as a visual representation of this “who’s who.”




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 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:

  • 1.You’ll start 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.
  • 2.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.
  • 3.Finally, you need your E. coli to take up that vector, in this case, the DE3 E. coli strain.

Inside E. coli

Inside the E.coli, which now contains your vector, the real action of 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:

  • 1.On 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.
  • 2.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.


Why Can’t I Use Lactose for Induction?

IPTG is structurally analogous to lactose, so why can’t either lactose or IPTG be used during induction? The answer is because IPTG is not part of the metabolic pathway. Because the cell doesn’t naturally use and break down IPTG, the concentration you begin with is going to remain relatively constant throughout the entire process. Therefore, the induction process is more efficient when using IPTG instead of lactose.



What is the Optimal IPTG Concentration to Use?

When it comes to the optimal optical density, the key is finding the point where all your cells are alive and very healthy (log phase or exponential phase), which is usually going to be an OD of around 0.6 – 0.8. When you get to an OD at higher levels, you run the risk of having dead cells that won’t be producing protein. Lower ODs than 0.6 – 0.8 are also not ideal because that just means you’re really working with more media than cells, and that will lead to lower expression.

The concentration of IPTG to use for expression is going to really come down to which plasmid you’re actually using. Because there are a few variables that can impact efficiency, it’s best to test on a few different colonies, using different ODs and IPTG concentrations.


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, from http://genetics.wustl.edu/tslab/protocols/protein-...

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, from https://www.ocf.berkeley.edu/~asiegel/posts/?p=22

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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