Construction and characterization of new host-vector systems for the enforcement-cloning method.
The Escherichia coli host strains, TH1, TH2, TH3 alpha, TH4 and TH5, all trpR-, rpsL- and supE-, were constructed to constitutively express a trp promoter/operator (POtrp)-driven synthetic rpsLam+ gene encoding the streptomycin sensitivity (Sms) determinant (ribosomal protein S12). The applicability of these strains to the Sms-enforcement cloning procedure [Toba-Minowa and Hashimoto-Gotoh, Gene 121 (1992) 25-33] was examined on tryptophan-rich low-salt (LS) agar medium in combination with two reconstructed Sms-enforcement plasmid vectors, ampicillin-resistant (ApR) pKF2, and chloramphenicol-resistant (CmR) pKF3. The results indicated that (1) pKF2 enforced the Sms phenotype on TH1, TH2, TH4 and TH5, but not TH3 alpha, while pKF3 was effective on all the strains, (2) even without Sm, strains TH1, TH2, TH4 and TH5 harboring pKF2 rarely formed colonies on LS+Ap agar, and (3) TH2 harboring pKF3 hardly grew, forming tiny colonies only after two overnight incubations at 37 degrees C on LS+Cm agar. By using the AseI, BclI, StuI and EcoRI sites in POtrp-rpsL+4am of pKF2 and pKF3, it was revealed that enforcement cloning was applicable in the new host-vector systems on normal nutrient agar medium, except for a combination of TH3 alpha and pKF2, with the TH2 strain in combination with pKF2 or pKF3 seeming to be most suitable for enforcement cloning, even without Sm.
Hashimoto-Gotoh
T
T
Department of Biochemistry and Molecular Genetics, Kyoto Prefectural University of Medicine, Japan.
Tsujimura
A
A
Kuriyama
K
K
Matsuda
S
S
D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000235:genetics; D001483:Base Sequence; D003001:Cloning, Molecular / Q000379:methods*; D004269:DNA, Bacterial; D004926:Escherichia coli / Q000235:genetics*; D029968:Escherichia coli Proteins; D005822:Genetic Vectors; D008969:Molecular Sequence Data; D009875:Operator Regions, Genetic; D010641:Phenotype; D010957:Plasmids; D011401:Promoter Regions, Genetic; D015183:Restriction Mapping; D000091243:Ribosomal Protein S9; D012269:Ribosomal Proteins / Q000235:genetics*
D001426:Bacterial Proteins; D004269:DNA, Bacterial; D029968:Escherichia coli Proteins; D000091243:Ribosomal Protein S9; D012269:Ribosomal Proteins; C000717190:RpsI protein, E coli; C034212:ribosomal protein S12