Glucose is the preferred food source for E. coli, however when glucose levels drop, E. coli need to look for other ways to feed themselves. One way in which they accomplish this is to replace glucose metabolism with lactose metabolism.
The induction and control of lactose metabolism is complicated and its process has been exploited by molecular biologists for different types of experiments. For example, the lac operon can be used to control gene expression. Additionally, genes from the lac operon can be used to bring a protein of interest to a particular piece of nuclei acid.
In this article, we’ll look at how the lac operon can be used to screen bacteria for recombinant plasmids. To understand the story, you need to know about two important aspects related to the lac operon.
Thank you for the compliment complement
The lac operon is a group of genes coding for the proteins that digest lactose in E. coli and some other enteric bacteria. How it works is explained in more detail in my first article.
The important gene in the lac operon to know about for today’s purposes is the lacZ gene, which codes for β-galactosidase (or more lovingly referred to as β-gal).
The β-gal enzyme functions in lactose digestion by cleaving lactose into glucose and galactose. In its active state, the enzyme exists as a homotetramer; four identical pieces associated with each other. Early on, a mutant β-gal enzyme derived from the M15 strain of E. coli was discovered that was missing a bunch of N-terminal residues. The absence of this chunk of protein leaves the enzyme unable to form a tetramer and thus it exists in an inactive state.
This mutant form was called the ω-peptide while the missing fragment was called the α-peptide.
Surprisingly it was found that enzyme activity is restored by supplying the α-peptide in trans, i.e. not linked to the protein. Thus, in the presence of its missing fragment, the enzyme works in a process termed α-complementation. Hold this thought in mind.
The other player in the story: IPTG
Like allolactose, IPTG or isopropyl-β-D-thio-galactoside, induces lac operon transcription. IPTG binds the lac repressor preventing it from inhibiting expression of the lac operon. Unlike allolactose, IPTG is not digested by β-galactosidase and so IPTG levels remain constant and the rate of lac operon expression does not fluctuate but occurs at predictable rates. This means that as you increase the concentration of IPTG added, lac operon expression will increase until it peaks and plateaus.
IPTG induction and blue/white screening
So you clone your DNA insert into a vector and transform it into bacteria. Every resulting colony should contain the recombinant plasmid, right? Unfortunately, this isn’t true and we can use a variety of tools to help us determine which colonies contain our plasmid of interest.
Blue/white screening is one of the earliest tools used to help identify the bacterial colony that contains your recombinant plasmid.
Here I am!
Imagine you are a state examiner and you’re trying to figure out which students have already received a copy of the test. The quickest way isn’t to check each student individually; it’s to ask everyone who hasn’t to raise his or her hand, effectively shouting “I do!”
Blue/white screening does exactly that! Well, not exactly per se. When plated on the appropriate medium (Xgal) colonies that have taken up the recombined plasmid containing your gene of interest remain colorless while the ones one who didn’t turn blue. It’d be cool if students could do that when you ask them yes or no questions and bacteria could raise tiny little hands to say no.
How they raise their hands
Blue/white screening works by disrupting α-complementation.
In blue/white screening, you use a host E. coli strain that contains the mutant lacZ gene encoding for the ω-peptide (i.e. β-gal inactive). The plasmid you are going to use contains a multiple cloning site within the sequences coding for the α-peptide. If you insert your sequence of interest correctly into the multiple cloning site, your sequence will disrupt expression of the α-peptide.
When transformed into E. coli, plasmids that do not contain an insert will provide the α-peptide and active β-gal will be expressed. Plasmids that contain your insert will not provide α-peptide and β-gal will remain inactive.
Active β-gal can be detected by X-gal. X-gal is a colorless analog of lactose (a.k.a. a lactose wannabe) that is cleaved by β-gal resulting in the formation of a bright blue pigment. If you plate your bacteria on agar containing X-gal, then any blue colonies have active β-gal, but don’t have your insert. You can collect the white colonies with more confidence that these bugs contain the plasmid with your insert.
Don’t forget the IPTG!
Even though this system sounds great, sometimes not enough β-gal is produced by the host cell to turn the colony blue. This is not useful to you because you will end up picking a bunch of useless bacteria.
To circumvent this problem, most people also add IPTG to the agar. The IPTG will derepress the lac operon, ensuring high expression of the mutant β-gal enzyme.
So that’s how you get rid of the cloning blues.