To investigate the factors influencing the artifical transformation in Escherichia coli E. coli C600 was transformed by pBR322 DNA with tetracycline and ampicillin resistant gene purified by CsCl-Etbr equilibrium density gradient centrifugation from E.coli HB 101.
The influencing factors in the transformation such as concentration of calcium chloride, time of ice incubation, temperature and time of heat shock, time of gene expression, effects of plasmid DNA concentration and adding time were examined in these experiments.
The results obtained were as follows;
1. The highest transformation frequency was observed in the treatments of 100 mM CaCl₂ before heat chock and the treatment of CaCl₂was essential step in the precess of E. coli transformation.
2. The highest transformation frequency was observed in the treatment of heat shock at 42℃ for 4 min. or 37℃ for: 6 min., but the prolonged heat shock resulted a decreased transformation frequency.
3. Treatments of ice incubation at 0℃ for 45 min. before heat stocks or at 0℃ for 30min. after heat shock resulted an increased transformation frequency.
4. There was a linear relationship between DNA concentration and transformation frequency ae the concentration of 8×10³ recipient cells. The highest transformation frequency reached in carte of 7 mcg of donor DNA, but above 1 mcg of DNA concentration, transformation frequency was not remarkably increased. Addition of donor DNA just after the treatment of CaCl₂ was the best.
5. The best condition of gene expression at 37℃ were 40min. for TC-resistant gene and 100min. for AP-resistant gene. TC-resistant gene was higher in the transformation frequency and faster in the gene expression time than AP-resistant gene.
In these results, the best conditions for the transformation of E. coli C 600 with pBR322 DNA were: treatment with 100mM CaCl₂, ice incubation at 0℃ for 45 min, heat shock at 42℃ for 4 min., 30 min. of ice incubation and incubation at 37℃ for 100min. for gene expression in that order.
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