Transformation inEscherichia coli: studies on the nature of donor DNA after uptake and integration

Abstract

Chromosomal E. coli DNA appears to be sensitive towards in vivo DNA restriction when transformed to a restrictive E. coli recipient. It is therefore concluded that transforming chromosomal donor DNA is present in a double-stranded form immediately after uptake.Genetic analysis of E. coli transformants, obtained with UV-irradiated donor DNA under conditions that exclude photorepair, show, especially in a uvrB recipient, loss of donor DNA information compared with the situation where DNA was not subjected to UV-irradiation. Similar conclusions were arrived at after genetic analysis of transductants obtained with UV-irradiated particles of the generalized transducing phage P i . The processing in E. coli of DNA after P i transduction is thus similar to that of transforming DNA. The observations are discussed and a possible explanation based on single-stranded DNA integration is presented in detail.

Treating Antibiotic Resistance Genes in Proteus Spp. were Isolated from Renal Stone Patients by Crataegus rhipidophylla and Adiantum capillus

“…] DNA uptake 100µl of prepared plasmid DNA (0.1) ml added to tube containing 0.2 ml of competent cells. The mixture was placed on ice for 30 minutes, exposed to heat shock at 42 C˚for 6 minutes [14]. After that 1 ml of fresh nutrient broth was added to transformation mixture, and incubated at 37C˚ for 60 minutes to allow the expression of the antibiotic resistant genes.…”

Cotransformation of linear chromosomal DNA and plasmid DNA in Escherichia coli

“…The procedure is a modification of one previously reported [7]. AM1268 was grown in phosphate-buffered minimal salts medium [12] till the absorbance reached a reading of 30 in a Klett photometer (660 nm filter).…”

“…When applied, the method would have the advantage that at no time do two, possibly interfering, plasmids have to be present in the same cell. The practical drawback that only recB recC sbcA/B strains are transformable with chromosomal DNA [5,6] can be circumvented since we have shown recently [7] that transformation of Rec ÷ strains with chromosomal DNA is possible, albeit at low frequencies.…”