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The wonders of lacZ E. coli

The process of colony selection can be simplified by choosing a vector and E.

colistrain that are compatible with blue/white colony screening. E. colistrains are

described as having a lacZ when they carry a mutation that deletes part of the -

galactosidase (lacZ) gene. The remaining portion of the gene is called the -

fragment. By using a plasmid that contains the deleted portion, or -fragment, the

function of the -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 -fragment. When

a sequence is inserted into this cloning region, the reading frame is disrupted, and

a non-functional -fragment is produced. This fragment is incapable of -

complementation. Growing the transformed bacteria on a plate containing 5-bromo-

4-chloro-3-indoyl- -D-galactopyranosidase (X-gal) will allow you to distinguish

between bacterial colonies formed from cells that contain plasmid with insert fromthose containing plasmid without insert. Any colony containing the plasmid (and

therefore the functioning -galactosidase gene) will turn blue, a result of the -

galactosidase activity. This is called -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 -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, DH5 and XL-1 Blue that have thenecessary 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 laclrepressor that stops transcription from the lac operon , and thus

production of the -fragment, until a substrate is present. The substrate, the non-

cleavable lactose analog, isopropyl--D-thiogalactopyranoside (IPTG), relieves the

repression of the lac operon and allows transcription to occur. These strains will

need to be grown on media 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 -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.

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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.