Beta-galactosidase
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Current revision (11:28, 10 June 2008) (view source) (links to protocols and X-Gal) |
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*A mutant with an internal deletion of codons 21-41 of the lacZ gene does not produce any active β-galactosidase. | *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. | *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 [http://www.cyto.purdue.edu/flowcyt/research/micrflow/jepras/jepras2.htm A flow cytometric study of stationary phase gene expression in E.coli using lacZ reporter gene fusion] | ||
| + | **This paper uses [http://probes.invitrogen.com/media/pis/mp02904.pdf C<sub>12</sub>FDG] from Invitrogen. However, FDG might be a better substrate in gram-negative bacteria like ''Escherichia coli'' <cite>Plovins-ApplEnvironMicrobio-1994</cite> | ||
| + | * Another fluorogenic substrate is 4-methylumbelliferyl β-D-galactopyranoside (MUG) which works in bacteria, yeast, and mammalian cells (without requiring permeabilization/lysis). <cite>Vidal-Aroca-2006</cite> | ||
| + | *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 <cite>Zamenhof-JBacteriol-1972</cite>. '''(If anyone has a better reference comparing results from a Miller assay of alpha-complementated β-galactosidase with wildtype, please include it here.)''' | ||
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| + | ==Protocols== | ||
| + | * [[beta-galactosidase assay]] | ||
| + | * [[LacZ staining of cells]] | ||
| + | |||
| + | ==See also== | ||
| + | * [[X-Gal]] | ||
==Reference== | ==Reference== | ||
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#Beckwith-1970 isbn=0317118099 | #Beckwith-1970 isbn=0317118099 | ||
#Ullmann-Bioessays-1992 pmid=1345751 | #Ullmann-Bioessays-1992 pmid=1345751 | ||
| + | #Plovins-ApplEnvironMicrobio-1994 pmid=7811104 | ||
| + | #Miller-1972 isbn=0879691069 | ||
| + | // original β-galactosidase assay by Miller | ||
| + | #Zamenhof-JBacteriol-1972 pmid=4552986 | ||
| + | #Thibodeau-Biotechniques-2004 pmid=15038156 | ||
| + | #WorthingtonBiochem http://www.worthington-biochem.com/BG/default.html | ||
| + | #Serebriiskii-AnalBiochem-2000 pmid=10998258 | ||
| + | #Vidal-Aroca-2006 pmid=16629389 | ||
</biblio> | </biblio> | ||
| - | [[Category:Material]] [[Category:Reporter]] | + | |
| + | [[Category:Material]] [[Category:Enzyme]] [[Category:Reporter]] | ||
Current revision
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
- 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.)
Protocols
See also
Reference
- Benno Muller-Hill. The Lac Operon. Walter de Gruyter. isbn:3-11-014830-7.
- isbn:0317118099.
- Plovins A, Alvarez AM, Ibañez M, Molina M, and Nombela C. . pmid:7811104.
- Vidal-Aroca F, Giannattasio M, Brunelli E, Vezzoli A, Plevani P, Muzi-Falconi M, and Bertoni G. . pmid:16629389.
- Zamenhof PJ and Villarejo M. . pmid:4552986.
- Ullmann A. . pmid:1345751.
- [by] Jeffrey H. Miller. Experiments in molecular genetics. [Cold Spring Harbor, N.Y.] Cold Spring Harbor Laboratory, 1972. isbn:0879691069.
original β-galactosidase assay by Miller - Thibodeau SA, Fang R, and Joung JK. . pmid:15038156.
- http://www.worthington-biochem.com/BG/default.html
- Serebriiskii IG and Golemis EA. . pmid:10998258.
