Construction of an ori cassette for adapting shuttle vectors for use in Haemophilus influenzae.
An ori (origin of DNA replication) cassette, pORC, containing the P15a ori and the kanamycin-resistance-encoding gene from Tn5, was constructed. The cassette was used to convert an Escherichia coli promoter selection vector, which gene from Tn5, was constructed. The cassette was used to convert an Escherichia coli promoter selection vector, which contains a promoterless chloramphenicol (Cm) acetyltransferase-encoding gene (cat) downstream from a multiple cloning site (MCS) [Brosius and Lupski, Methods Enzymol. 153 (1987) 54-68], to an E. coli-Haemophilus influenzae shuttle vector. The shuttle vector, pQL1, was shown to transform E. coli and H. influenzae efficiently. H. influenzae promoters were cloned into pQL1 by ligation of Sau3A-digested H. influenzae chromosomal fragments. Selection and semiquantitative analysis of promoter strength were performed on agar plates containing different concentrations of Cm. With the use of pQL1, H. influenzae gene regulation can now be studied in either H. influenzae or E. coli. In addition, elements of pORC can be used to convert other specialized E. coli vectors to E. coli-H. influenzae shuttle vectors.
Department of Pediatrics, Medical College of Pennsylvania, Philadelphia 19129.
G J
GJ
Heidecker
J M
JM
Pozsgay
T L
TL
Stull
D001483:Base Sequence; D003001:Cloning, Molecular; D017931:DNA Primers; D004269:DNA, Bacterial; D004926:Escherichia coli / Q000235:genetics; D005069:Evaluation Studies as Topic; D015870:Gene Expression; D005822:Genetic Vectors; D006193:Haemophilus influenzae / Q000235:genetics*; D008969:Molecular Sequence Data; D018741:Replication Origin / Q000235:genetics*; D014169:Transformation, Bacterial
D017931:DNA Primers; D004269:DNA, Bacterial