APPLICATION OF GALACTOSE-SENSITIVE ESCHERICHIA-COLI STRAINS AS SELECTIVE HOSTS FOR LACZ- PLASMIDS
APPLICATION OF GALACTOSE-SENSITIVE ESCHERICHIA-COLI STRAINS AS SELECTIVE HOSTS FOR LACZ- PLASMIDS
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DOI：
10.1093/nar/20.12.3254
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发表时间：
1992-06-25
影响因子：
14.9
通讯作者：
VIJG, J
中科院分区：
文献类型：
--
作者：
GOSSEN, JA;MOLIJN, AC;VIJG, J
E. coli strains carrying a point mutation in the GalE gene are highly sensitive to galactose (1). Due to inactive UDP-epimerase, such strains are unable to convert UDP-galactose into UDP-glucose, resulting in cell lysis during early cell growth. Since the, B-galactosidase enzyme, encoded by the LacZ gene, converts lactose into galactose we reasoned that GalE-strains should be applicable to select for colonies harbouring LacZ-plasmids. To develop an assay system in which LacZ-cells are unable to grow, a galactose sensitive E. coli C strain was made. The GalE-gene from strain PC3415 was transferred to a LacZ-E. coli C strain (2) by means of P1 transduction. Galactose-sensitivity was tested by transforming E. coli C (LacZ-/GalE-) with the LacZ-containing plasmid pUR288. Cells were plated on LB/M9 minimal agar medium (1: 9 vol/vol; ref. 3) containing 50 rig/ml ampicillin and 0.05% phenylgalactoside (Sigma) and incubated overnight at 37'C. LB agar medium containing 50 p4g/ml ampicillin and 0.3 atg/mi Xgal was used as a control medium. The results obtained (Table 1), show that E. coli C (LacZ-/GalE-) cells harbouring the pUR288 plasmid are unable to grow on LB/M9 minimal agar medium in the presence of 0.05% phenylgalactoside. E. coli C (LacZ-/GalE-) harbouring the pBR322 (LacZ-) plasmid, however, are not affected (Table 1). The same results were obtained for E. coli C (LacZ-/GalE-) containing eitherthe pUR288 or pBR322 plasmid when plated on control LB agar medium. To test whether such a selection system would be able to reproducibly detect low numbers of LacZ-E. coli cells among large numbers of LacZ+ E. coli cells, different mixtures of LacZ+/LacZ-cells were plated on selective and non-selective medium. The results, as shown in Table 2, indicate that LacZ-colonies are easily detectable, even in the presence of a 105 fold excess of LacZ+ colonies. In conclusion, the use of galE-E. coli strains enables selection for LacZ-plasmids. One application of GalE-strains would be their use as hosts in cloning experiments. Most commonlyused cloning vectors contain a multiple cloning site within the LacZc gene. After transformation to E. coli host cells, recombinant plasmids are recognized on basis of the absence of cxcomplementation. Since, 3-galactosidase expression will be toxic when GalE-cells are grown on LB/M9 minimal medium in the presence of phenylgalactoside, these strains would allowselective growth of plasmids containing inserts only. Time-consuming steps like dephosphorylation of vectors and the use of X-gal/IPTG indicator plates can therefore be omitted. A second application of GaIE-strains is in mutation studies employing LacZ as the mutational target gene (2). Using transgenic mice with integrated plasmids, containing the LacZ marker gene (4), determination of mutation frequencies no longer requires the analysis of large numbers of rescued vectors but can simply be determined as the ratio of the number of colonies on LB/M9 medium versus the number of colonies on LB medium.
