APPENDIX 5: X-gal AND IPTG

Use of X-gal

X-gal (5-bromo-4-chloro-indolyl-fl-D-galactoside) is a chromogenic

substrate for/3-galactosidase. When X-gal is cleaved by/3-galactosidase,
the blue 5-bromo-4-chloro indigo is produced. X-gal is used in plates at
a concentration of 40/zg/ml. X-gal is soluble in dimethyl formamide. The
amount of X-gal needed is dissolved in the smallest volume of dimethyl
formamide possible and then added to agar media before plates are poured.

Use of IPTG

IPTG (isopropyl-/3-D-thiogalactoside) is an inducer of the lac operon.

IPTG is water soluble and is used at 120/zg/ml. The lac operon consists
of a promoter, P, an operator, O, and the lacZ (/3-galactosidase), lacy
(permease), and lacA (acetylase) genes. The lacI gene encodes a repressor
protein that binds the operator and prevents transcription of the lac opo
eron. For transcription of the lac operon to occur, the repressor cannot be
bound to the operator. In addition, a positive regulator, a complex of cyclic
AMP and catabolic activating protein, is required for RNA polymerase
binding to the promoter.
The Lac repressor protein interacts with IPTG and no longer binds
the operator region of the lac operon. Also note that in the absence of
glucose, cyclic AMP is produced and interacts with the catabolic activating
protein to form a complex that binds to the promoter of the lac operon to
allow RNA polymerase to transcribe the lac operon. Thus, even if IPTG

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248 APPENDIX 5: X~al AND IPTG

were present, if glucose were also present in the medium, the lac operon
would not be expressed.

pUC Vectors and Need for IPTG

Note that pUC cloning vectors contain lacI' (Hackett et al., 1984).
lacI' refers to only a piece of the lacI gene. pUC contains part of the
lac regulatory region~the operator and promoter but not a functional
repressor. When cloning into pUC vectors is being performed and pUC is
being introduced into E. coli strains, the genotype of the E. coli strain
should be examined. The recipient E. coli strain should be lac-. When
cloning into strains such as DH5a is being performed, the inducer IPTG
is not needed in the plates. Also check to see if the strain is lacIq, lacIq is
a mutation that causes an overproduction of the lac repressor protein. To
allow transcription of the lac operon in a lacIq strain, an inducer (IPTG)
must be added. The lacIq strain is useful to regulate the transcription of
a gene cloned into the pUC polylinker.

Reference

Hackett, P. B., Fuchs, J. A., and Messing, J. W. (1984). "An Introduction to Recombinant
DNA Techniques: Basic Experiments in Gene Manipulation." 1st Ed., p. 38. Benjamin/
Cummings, New York.