Construction and characterization of a novel two-plasmid system for accomplishing temperature-regulated, amplified expression of cloned adventitious genes in Escherichia coli.
We describe a two-plasmid system that utilizes the lacZ gene promoter and temperature-responsive plasmid replicons to accomplish closely regulated high-level expression of heterologous genes in Escherichia coli. One of the plasmids fails to replicate at 42 degrees C and contains a gene encoding the lac repressor; the second plasmid, which undergoes multicopy "runaway" replication at elevated temperatures, contains an adventitious gene under control of the operator-promoter system of the lacZ gene. Concurrent derepression of lac promoter function and amplification of copy number of the lac-controlled gene occurs when the temperature is elevated. We have used a structural gene encoding chloramphenicol acetyltransferase to demonstrate that the gene product under control of the lacZ promoter represents a major fraction of the total protein synthesized at 43 degrees C, whereas only minimal quantities of this enzyme are made at 30 degrees C. The system described allows the controlled expression of gene products that may have detrimental effects on cell growth, and provides a simple method for identifying radioactivity-labeled protein products of cloned genes in bacterial whole-cell extracts. The system also offers an alternative to intragenic temperature-sensitive mutations for studying the function of various enzymatic or regulatory proteins.
D000123:Acetyltransferases / Q000235:genetics; D015500:Chloramphenicol O-Acetyltransferase; D003001:Cloning, Molecular; D004274:DNA, Recombinant / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics*; D005784:Gene Amplification; D007763:Lac Operon; D010957:Plasmids; D012107:Research Design; D013696:Temperature
D004274:DNA, Recombinant; D000123:Acetyltransferases; D015500:Chloramphenicol O-Acetyltransferase