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Construction and characterization of new host-vector systems for the enforcement-cloning method
T Hashimoto-Gotoh1, A Tsujimura, K Kuriyama
1Department of Biochemistry and Molecular Genetics, Kyoto Prefectural University of Medicine, Japan.
Gene
|December 31, 1993
Summary
New Escherichia coli strains and plasmid vectors were developed for streptomycin sensitivity (Sms) enforcement cloning. The TH2 strain with either pKF2 or pKF3 plasmid proved most effective for this cloning method.
Area of Science:
- Molecular Biology
- Microbiology
- Bacterial Genetics
Background:
- Development of novel Escherichia coli host strains (TH1-TH5) engineered for constitutive expression of a streptomycin sensitivity (Sms) determinant.
- These strains carry a trp promoter/operator (POtrp)-driven synthetic rpsLam+ gene encoding ribosomal protein S12, conferring streptomycin sensitivity (Sms).
Purpose of the Study:
- To evaluate the applicability of newly constructed Escherichia coli strains and plasmid vectors for the streptomycin sensitivity (Sms)-enforcement cloning procedure.
- To identify optimal host-vector combinations for efficient enforcement cloning.
Main Methods:
- Construction of five Escherichia coli host strains (TH1-TH5) with specific genetic modifications (trpR-, rpsL-, supE-).
- Utilization of two Sms-enforcement plasmid vectors: ampicillin-resistant (ApR) pKF2 and chloramphenicol-resistant (CmR) pKF3.
- Testing strain-vector compatibility on tryptophan-rich low-salt (LS) agar medium and standard nutrient agar, assessing colony formation and growth.
Main Results:
- Plasmid pKF2 successfully enforced the Sms phenotype on strains TH1, TH2, TH4, and TH5, but not TH3 alpha; pKF3 was effective on all tested strains.
- Strains TH1, TH2, TH4, and TH5 with pKF2 showed limited colony formation on LS+Ap agar even without streptomycin (Sm).
- Strain TH2 with pKF3 exhibited restricted growth on LS+Cm agar, forming tiny colonies after extended incubation.
- Enforcement cloning was found applicable across most new host-vector systems on normal nutrient agar, with TH2 and pKF2 or pKF3 being particularly suitable.
Conclusions:
- The developed Escherichia coli host strains and plasmid vectors are largely applicable to the Sms-enforcement cloning procedure.
- The TH2 strain, in combination with either pKF2 or pKF3, demonstrates significant suitability for enforcement cloning, even in the absence of streptomycin.
- Further optimization of specific host-vector combinations may be necessary for certain applications, as highlighted by the TH3 alpha and pKF2 incompatibility.