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Some interesting facts [1]

  • A tetramer of 4 identical subunits
  • Each subunit is 120kD.
  • Active only as a tetramer.
  • Mutations in some of the codons of the N-terminal 60aa or C-terminal 100aa results in an inactive, dimeric β-galactosidase. [2]
  • If the above sequences are deleted, the missing protein fragment can be replaced by the corresponding peptide. This is called intracistronic alpha or omega complementation respectively.
  • Its N-terminal 23 residues can be replaced by any amino acid residues without affecting the enzymatic activity.
  • A mutant with an internal deletion of codons 21-41 of the lacZ gene does not produce any active β-galactosidase.
  • A mutant with a deletion of everything past residue 60 (i.e. it expresses only the first 60 N-terminal amino acids) does not produce any active β-galactosidase.
  • You can measure lacZ activity using flow cytometry. See A flow cytometric study of stationary phase gene expression in E.coli using lacZ reporter gene fusion
    • This paper uses C12FDG from Invitrogen. However, FDG might be a better substrate in gram-negative bacteria like Escherichia coli [3]
  • Another fluorogenic substrate is 4-methylumbelliferyl β-D-galactopyranoside (MUG) which works in bacteria, yeast, and mammalian cells (without requiring permeabilization/lysis). [4]
  • Alpha-complementation of β-galactosidase does not seem to yield activities equal to wildtype β-galactosidase. Depending on the fragment, the activity can be up to 24% of wildtype [5]. (If anyone has a better reference comparing results from a Miller assay of alpha-complementated β-galactosidase with wildtype, please include it here.)


See also


  1. ISBN:3-11-014830-7 [Muller-Hill-1996]
  2. ISBN:0317118099 [Beckwith-1970]
  3. Plovins A, Alvarez AM, Ibañez M, Molina M, and Nombela C. Use of fluorescein-di-beta-D-galactopyranoside (FDG) and C12-FDG as substrates for beta-galactosidase detection by flow cytometry in animal, bacterial, and yeast cells. Appl Environ Microbiol. 1994 Dec;60(12):4638-41. DOI:10.1128/aem.60.12.4638-4641.1994 | PubMed ID:7811104 | HubMed [Plovins-ApplEnvironMicrobio-1994]
  4. Vidal-Aroca F, Giannattasio M, Brunelli E, Vezzoli A, Plevani P, Muzi-Falconi M, and Bertoni G. One-step high-throughput assay for quantitative detection of beta-galactosidase activity in intact gram-negative bacteria, yeast, and mammalian cells. Biotechniques. 2006 Apr;40(4):433-4, 436, 438 passim. DOI:10.2144/000112145 | PubMed ID:16629389 | HubMed [Vidal-Aroca-2006]
  5. Zamenhof PJ and Villarejo M. Construction and properties of Escherichia coli strains exhibiting -complementation of -galactosidase fragments in vivo. J Bacteriol. 1972 Apr;110(1):171-8. DOI:10.1128/jb.110.1.171-178.1972 | PubMed ID:4552986 | HubMed [Zamenhof-JBacteriol-1972]
  6. Ullmann A. Complementation in beta-galactosidase: from protein structure to genetic engineering. Bioessays. 1992 Mar;14(3):201-5. DOI:10.1002/bies.950140311 | PubMed ID:1345751 | HubMed [Ullmann-Bioessays-1992]
  7. ISBN:0879691069 [Miller-1972]

    original β-galactosidase assay by Miller

  8. Thibodeau SA, Fang R, and Joung JK. High-throughput beta-galactosidase assay for bacterial cell-based reporter systems. Biotechniques. 2004 Mar;36(3):410-5. DOI:10.2144/04363BM07 | PubMed ID:15038156 | HubMed [Thibodeau-Biotechniques-2004]
  9. [WorthingtonBiochem]
  10. Serebriiskii IG and Golemis EA. Uses of lacZ to study gene function: evaluation of beta-galactosidase assays employed in the yeast two-hybrid system. Anal Biochem. 2000 Oct 1;285(1):1-15. DOI:10.1006/abio.2000.4672 | PubMed ID:10998258 | HubMed [Serebriiskii-AnalBiochem-2000]

All Medline abstracts: PubMed | HubMed