A novel cloning system for direct screening using a suicidal strategy.
The pAC92 plasmid is a direct screening cloning vector which allows positive selection of recombinant clones (re-clones). This new high-copy-number plasmid vector encodes ampicillin resistance and carries the Bacillus subtilis alpha-amylase (alpha-Amy)-encoding gene (amy) containing a multiple cloning site. The pAC92 plasmid confers to Escherichia coli transformants an amylolytic phenotype easily detected by iodine vapor staining. The re-clones are identified by insertional inactivation of alpha-Amy activity. During pAC92 construction, a bacterial growth defect was observed in host cells after some modifications of the promoter region that caused the increase in the amy expression. This suicide characteristic permitted the positive selection of re-clones. A second transformation step was performed to enhance the rate of re-clones per plate.
Barros
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EV
Laboratory of Molecular Biology, Department of Cellular Biology, University of Brasilia, Brazil.
Bataus
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LA
Valência
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Maranhão
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AQ
Astolfi-Filho
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D001412:Bacillus subtilis / Q000201:enzymology / Q000235:genetics; D001483:Base Sequence; D003001:Cloning, Molecular / Q000379:methods*; D004274:DNA, Recombinant; D004926:Escherichia coli / Q000235:genetics / Q000254:growth & development; D005822:Genetic Vectors; D008969:Molecular Sequence Data; D013194:Staining and Labeling; D000516:alpha-Amylases / Q000235:genetics*
D004274:DNA, Recombinant; D000516:alpha-Amylases