Measurement of cat expression from growth-rate-regulated promoters employing beta-lactamase activity as an indicator of plasmid copy number.
Many promoter-fusion vectors contain an intact beta-lactamase (BLA) gene (bla) to allow measurement of BLA activity as an internal control for plasmid copy number. This approach rests on the assumption that bla is constitutively expressed. To use such vectors for comparison of promoter activity at different growth rates it was necessary to confirm that this is the case under all physiological conditions. The relationship between plasmid copy number and BLA activity at different steady-state growth rates in Escherichia coli HB101 transformed with a ColE1-type plasmid (pBR325) was examined. Both BLA activity and plasmid copy number decreased in a parallel fashion as growth rate increased. This finding was tested further by measuring the growth-rate-regulated expression of the chloramphenicol acetyltransferase (CAT) gene under the control of the rrnB P1 promoter in a plasmid pKK231-1 fusion. The results indicate that BLA activity is a reliable indicator of copy number at a wide range of growth rates and that CAT/BLA ratios can be employed as a valid measure of promoter-specific activity in such plasmid fusions under these different physiological conditions.
Klotsky
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RA
Schwartz
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I
D000123:Acetyltransferases / Q000235:genetics* / Q000378:metabolism; D002455:Cell Division; D015500:Chloramphenicol O-Acetyltransferase; D004261:DNA Replication; D004926:Escherichia coli / Q000235:genetics; D005786:Gene Expression Regulation; D005822:Genetic Vectors; D010957:Plasmids; D011401:Promoter Regions, Genetic; D012335:RNA, Ribosomal / Q000235:genetics; D001618:beta-Lactamases / Q000235:genetics* / Q000378:metabolism
D012335:RNA, Ribosomal; D000123:Acetyltransferases; D015500:Chloramphenicol O-Acetyltransferase; D001618:beta-Lactamases