实验概要
α-complementation occurs when two inactive fragments of E. coli β-galactosidase associate to form a functional enzyme. Many plasmid vectors carry a short segment of DNA containing the coding information for the first 146 amino acids of β-galactosidase. Vectors of this type are used in host cells that express the carboxy-terminal portion of the enzyme. Although neither the host nor the plasmid-encoded fragments of β-galactosidase are themselves active, they
can associate to form an enzymatically active protein. Lac bacteria that result from α-complementation are easily recognized because they form blue colonies in the presence of the chromogenic substrate X-gal. However, insertion of a fragment of foreign DNA into the polycloning site of the plasmid almost invariably results in production of an aminoterminal fragment that is no longer capable of α-complementation. Bacteria carrying recombinant plasmids therefore form white colonies. The development of this simple blue-white color test has greatly simplified the identification of recombinants constructed in plasmid vectors.
实验材料
Buffers and Solutions
IPTG solution (20% w/v)
X-gal solution (2% w/v)
Media
Rich broth agar plates containing the appropriate antibiotic
Rich broth top agar
Vectors and Bacterial Strains
E. coli culture, transformed with recombinant plasmids
实验步骤
1. Dispense aliquots of molten top agar into 17 x 100-mm tubes. Place the tubes in a 45°C heating block until they are needed.
2. Remove the first tube from the heating block. Working quickly,
add 0.1 ml of bacterial suspension containing <3000 viable bacteria
for a 90-mm plate and
<10,000 for a 150-mm plate. Close the
top of the tube and invert it several times to disperse the bacteria
through the molten agar.
3. Open the tube and add the appropriate amounts of X-gal and IPTG
(if required) as shown in the table below. Close the top of the tube and
gently invert it
several times to mix the contents.
Components for Top Agar
Amount of Reagent
Size of Plate Molten Top Agar X-gal IPTG
90 mm 3 ml 40 μl 7 μl
150 mm 7 ml 100 μl 20 μl
IPTG May not be required; please see the entry on IPTG in the Materials list.
4. Quickly pour the molten top agar into the center of a hardened agar plate containing the appropriate antibiotic and distribute the solution by swirling.
5. Repeat Steps 2-4 until all of the samples have been plated.
6. Allow the soft agar to harden at room temperature, wipe any
condensation from the lid of the plates, and then incubate the plates in
an inverted position for
12-16 hours at 37°C.
7. Remove the plates from the incubator and store them for several hours at 4°C, to allow the blue color to develop.
8. Identify colonies carrying recombinant plasmids.
• Colonies that carry wild-type plasmids contain active -galactosidase. These colonies are pale blue in the center and dense blue at their periphery.
• Colonies that carry recombinant plasmids do not contain
active -galactosidase. These colonies are creamy-white or eggshell blue,
sometimes with a faint blue
spot in the center.
9. Select and culture colonies carrying recombinant plasmids.