Chitobiase, a new reporter enzyme

N,N'-diacetylchitobiase (chitobiase) from the marine organism Vibrio harveyi is a highly stable reporter enzyme for gene fusions. This enzyme hydrolyzes the disaccharide chitobiose to N-acetyl glucosamine. The advantages of the reporter gene encoding chitobiase (chb) are: (i) that chitobiase an...

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Veröffentlicht in:BioTechniques. - 1993. - 25(1998), 6 vom: 04. Dez., Seite 1030-5
1. Verfasser: Kalabat, D Y (VerfasserIn)
Weitere Verfasser: Froelich, J M, Phuong, T K, Forsyth, R A, Newman, V G, Zyskind, J W
Format: Aufsatz
Sprache:English
Veröffentlicht: 1998
Zugriff auf das übergeordnete Werk:BioTechniques
Schlagworte:Journal Article Research Support, U.S. Gov't, Non-P.H.S. Research Support, U.S. Gov't, P.H.S. Bacterial Proteins DNA-Binding Proteins DnaA protein, Bacteria Recombinant Fusion Proteins Acetylglucosaminidase EC 3.2.1.52
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245 1 0 |a Chitobiase, a new reporter enzyme 
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520 |a N,N'-diacetylchitobiase (chitobiase) from the marine organism Vibrio harveyi is a highly stable reporter enzyme for gene fusions. This enzyme hydrolyzes the disaccharide chitobiose to N-acetyl glucosamine. The advantages of the reporter gene encoding chitobiase (chb) are: (i) that chitobiase and N-acetyl-beta-D-glucosaminidase activities are missing in E. coli strains, (ii) chitobiase can be monitored using blue/white colony indicator plates and (iii) convenient substrates for this enzyme are commercially available. The use of chitobiase as a reporter enzyme is generally applicable to the study of gene expression in those bacteria that do not contain N-acetyl-beta-D-glucosaminidases. We constructed plasmid vectors containing a multiple cloning site for producing in-frame fusions to chitobiase, the attP of lambda phase for movement into the bacterial chromosome for single-copy analysis, the gene encoding chloramphenicol acetyltransferase (cat), the pACYC184 origin of replication and the rrnBt1t2 terminator region upstream of the chb gene to prevent read-through from other promoters. In-frame fusions between the dnaA gene and chb were moved to the chromosome by site-specific recombination with the chromosomal attB site. These single-copy fusions were assayed for chitobiase to examine the effects of a deletion in the dnaA regulatory region 
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650 4 |a Research Support, U.S. Gov't, P.H.S. 
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650 7 |a DNA-Binding Proteins  |2 NLM 
650 7 |a DnaA protein, Bacteria  |2 NLM 
650 7 |a Recombinant Fusion Proteins  |2 NLM 
650 7 |a Acetylglucosaminidase  |2 NLM 
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700 1 |a Phuong, T K  |e verfasserin  |4 aut 
700 1 |a Forsyth, R A  |e verfasserin  |4 aut 
700 1 |a Newman, V G  |e verfasserin  |4 aut 
700 1 |a Zyskind, J W  |e verfasserin  |4 aut 
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