Ah, the wonders and frustrations of cloning. We’ve all been there. After careful planning, you have created the cloned plasmid containing your DNA sequence of interest, transformed it into bacterial cells and carefully spread those cells on a plate to grow. Now you stand at your bench gazing down at your master piece: a plate full of tiny bacterial colonies. Somewhere inside those cells is your DNA sequence, happily replicating with its plasmid host. But wait – logic tells you that not ALL of those colonies can contain your plasmid. There must be hundreds of colonies. Which ones have your plasmid? You begin to panic. Visions of yourself old and grey and still screening colonies flash through your mind. At the next bench, your lab-mate is cheerfully selecting colonies to screen. Although there are hundreds of colonies on her plate as well, some are white and some are blue. She is only picking the white colonies. What does she know that you don’t?
The wonders of lacZΔ E. coli
The process of colony selection can be simplified by choosing a vector and E. coli strain that are compatible with blue/white colony screening. E. coli strains are described as having a lacZΔ when they carry a mutation that deletes part of the β-galactosidase (lacZ) gene. The remaining portion of the gene is called the ω-fragment. By using a plasmid that contains the deleted portion, or α-fragment, the function of the β-galactosidase gene can be restored once the plasmid has been incorporated into the bacterium. For blue/white colony screening, the plasmids have a multiple cloning region within the coding sequence of the α-fragment. When a sequence is inserted into this cloning region, the reading frame is disrupted, and a non-functional α-fragment is produced. This fragment is incapable of α-complementation. Growing the transformed bacteria on a plate containing 5-bromo-4-chloro-3-indoyl-β-D-galactopyranosidase (X-gal) will allow you to distinguish between bacterial colonies formed from cells that contain plasmid with insert from those containing plasmid without insert. Any colony containing the plasmid (and therefore the functioning β-galactosidase gene) will turn blue, a result of the β-galactosidase activity. This is called α-complementation. Those colonies containing plasmids with an insert can be differentiated from those without an insert by the color of the colony (white versus blue). The insert disrupted the β-galactosidase gene, and therefore these colonies remain white. Colonies that did not pick up any plasmid at all will also appear as white colonies; however, most plasmids contain an antibiotic resistance gene that can be used for selection (see below).
There are a number of strains including JM109, DH5α and XL-1 Blue that have the necessary deletions and can be used for blue/white colony screening. However, the mechanism for blue/white screening is slightly different for JM109 and XL-Blue. Both of these strains also have a second mutation, laclq, which increases production of the lacl repressor that stops transcription from the lac operon , and thus production of the α-fragment, until a substrate is present. The substrate, the non-cleavable lactose analog, isopropyl-β-D-thiogalactopyranoside (IPTG), relieves the repression of the lac operon and allows transcription to occur. These strains will need to be grown on medium containing IPTG as well as X-gal.
Occasionally, colonies will appear pale blue, not white. As long as you see colonies on your plate that are darker blue, try picking some of the pale blue colonies, chances are good that they have the constructed plasmid that contains your DNA fragment.
In addition to the β-galactosidase marker, most cloning plasmids will also contain a gene that confers resistance to an antibiotic such as ampicillin. Using Ampicillin (or other appropriate antibiotic) in your growth medium should prevent bacteria that did not take up the plasmid during the transformation from growing. This way you can be fairly confident that the white colonies you see on your screening plate contain plasmid with insert.
And of course it is always a good idea to run controls with your cloning experiment. A plasmid-only control should give you a plate of blue colonies, and this will let you know that your transformation worked. To make sure that the antibiotic in your selective medium is effective, plate some untransformed cells. Few, if any, colonies should be observed on this plate.
Want to learn more and become a true rock star at subcloning? Download the Subcloning Notebook.
Looking for a quick and easy way to count colonies on agar plates? Download the Promega Colony Counter app. Take a picture of a plate, and the colonies are automatically counted within seconds. Results can be quickly refined by marking additional colonies and masking reflections.
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