Plasmid GENE Trasfer Invitro into Auxotroph Pheno-Genotypes, F-, Escherichia coli K-12, Generate Hybrid Prototrophs to  Damage Clonal Selectivity

After the discovery of DNA double helix in 1953 by Watson and Crick, the concepts of DNAà RNAàProtein, the dogma of life was more or less clear and the concepts of recipients and donor specially in microbial systems predominated to study jumping gene (transposable= Tn) element, IS1sequences, involved in plasmid recombination and antibiotic resistance. Slowly this concept was moved to design vaccine and for the productions of hormones (insulin, growth hormones, interleukin) (i.e., to develop genetically engineered hybrid Escherichia coli ) that would express and help many such fundamental aspects of genetic engineering and molecular biology. In such design environment with microbes, some basic important items were included, it was essential; to know the functions of the donor (i.e. the microbes, those are industrially important), whether they posses, plasmids or not; to know the recipient( i.e the universal recipient, that accepts foreign DNA, the well known  Escherichia coli, K-12, the Yale strain. The strain was isolated through various mutations, caused by X-ray and UV, and was compatible to accept the foreign DNA and to help to express at K-12 auxotrophic pheno-genotypes. After that the concept of selectivity of specific DNA in transforming recipient (F-) through plasmid DNA vectors took leading part. pBR322, PSU101, and other plasmid vectors were developed. All these plasmid DNA vectors have small DNA sequence (i.e. length less than 3 Kb) and carry operon (OP) (operator and promoter sequences) and Ori( Origin of replication), support clone DNA to express at auxoytophic pheno-geno types. In all such cases the selectivity in recipient ( F-) Escherichia coli K-12  at auxotrophs was challenged( i.e it will grow in minimal agar, only in presence of amino acids(leucien, methionine, tryptophan). These amino acids were damaged in mutations. In addition to that some antibiotic resistant markers were added, that perhaps was not present in vector plasmid and cloned targeted DNA. Combined antibiotics selectivity allowed growing the selective clone on minimal agar plates. Beyond in vitro plasmid transformation, in vivo some self designed co-conjugation process were applied (i.e allowing the donor plasmid to express at autotrophic phenol-genotypes). However in most of all recombination, r-DNA technology of plasmids lead to prototroph. In this paper the author will show one of such experimental steps with MRHU (D-mannose resistant haemagglutination of human erythrocyte), a gene that indirectly signifies the colonization and adherence factor of fatal pathogenic activities of Escherichia coli. At the frequency of 10^{- 3} a hybrid 5405 clone was selected, expressing MRHU(+) factor of the donor at auxotrophic phenol-geno types and was used for Balb/c mice immune response against adherence and fatal pathogenic activities of 026:EPEC ( Enteropathogenic Escherichia coli).