A recombination system has been developed for efficient chromosome engineering in Escherichia coli by using electroporated linear DNA. A defective lambda prophage supplies functions that protect and recombine an electroporated linear DNA substrate in the bacterial cell. The use of recombination eliminates the requirement for standard cloning as all novel joints are engineered by chemical synthesis in vitro and the linear DNA is efficiently recombined into place in vivo. The technology and manipulations required are simple and straightforward. A temperature-dependent repressor tightly controls prophage expression, and, thus, recombination functions can be transiently supplied by shifting cultures to 42 degrees C for 15 min. The efficient prophage recombination system does not require host RecA function and depends primarily on Exo, Beta, and Gam functions expressed from the defective lambda prophage. The defective prophage can be moved to other strains and can be easily removed from any strain. Gene disruptions and modifications of both the bacterial chromosome and bacterial plasmids are possible. This system will be especially useful for the engineering of large bacterial plasmids such as those from bacterial artificial chromosome libraries.

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An efficient recombination system for chromosome engineering in Escherichia coli

https://mmbr.asm.org/content/mmbr/36/4/525.full.pdf

Pedigrees of some mutant strains of Escherichia coli K-12.

https://mmbr.asm.org/content/mmbr/48/1/60.full.pdf

Mutagenesis and inducible responses to deoxyribonucleic acid damage in Escherichia coli.

INTRODUCTION .............................................................. 869 Enzymatic Repair of UV Damage in E. coli ......... .......................... 869 Error-Proof and Error-Prone Pathways of DNA Repair ....... ................. 871 The \"SOS\" Hypothesis: the Regulatory Role of DNA Damage ...... ............ 874 EVIDENCE FOR THE INDUCIBILITY OF ERROR-PRONE REPAIR (\"SOS REPAIR\") ACTIVITY ..................................................... 875 SOS Repair of Bacteriophage DNA ............ ............................... 875 SOS Repair of Bacterial DNA ................ ................................ 876 Evidence from studies of repair-deficient mutants ....... .................... 876 Evidence from studies of postreplication repair ........ ...................... 879 MANIFESTATIONS OF SOS REPAIR AND THEIR SIGNIFICANCE ..... ...... 879 Mutation Frequency Response to Increasing Fluence of UV Radiation ..... ..... 879 The UV Lesion Responsible for Induction of SOS Functions.................... 880 Kinetics of Induction and Decay of SOS Repair Activity ....... ................ 882 Time of Action of SOS Repair in UV Mutagenesis ...... ........................ 882 Cryptic Premutational Lesions Susceptible to SOS Repair ...................... 884 Mutator Effect of SOS Repair on Undamaged DNA ....... ..................... 885 PLASMIDS AND SOS REPAIR ................................................ 886 MECHANISM OF SOS REPAIR ............................................... 886 Mechanism of SOS Repair in Bacteriophage ......... .......................... 886 Mechanism of SOS Repair in Bacteria ........... ............................. 887 IMPLICATIONS OF SOS REPAIR FOR CARCINOGENESIS ................... 888 REGULATION OF SOS REPAIR AND OTHER UV-INDUCIBLE FUNCTIONS . 889 PROTEASES AND THE EXPRESSION OF SOS FUNCTIONS .................. 894 CONCLUSIONS ............................................................... 895 APPENDIX................................................................... 896 LITERATURE CITED ......................................................... 898

Ultraviolet mutagenesis and inducible DNA repair in Escherichia coli.

Biochemistry of homologous recombination in Escherichia coli.

The behavior of lambda phage in the Rec(-) strain JC-1569 is compared with that in the Rec(+) strain JC-1557. No difference deemed significant was noted in the adsorption rate, latent period, burst size, frequency of lysogenization, and frequency of vegetative phage recombination. The location of the prophage and its mode of insertion in the Rec(-) lysogen of wild-type lambda (lambda(+)) were inferred to be normal from the results of conjugational crosses. Spontaneous and ultraviolet (UV) irradiation induction of lambda(+) were markedly reduced in the Rec(-) lysogen. On the other hand, thermal induction of a mutant lambda (lambdacI857) lysogen of the Rec(-) strain was not reduced and was only slightly affected by UV irradiation. Phage subject to inhibition by lambda immunity failed to multiply in UV-irradiated cells of the Rec(-) lambda(+) lysogen, whereas those not inhibited by this immunity did multiply. It was concluded that the failure of UV to induce lambda(+) in the Rec(-) lysogen was not due to damage to the prophage, but rather to the inability of the irradiated cells to respond by lifting immunity. Preliminary evidence indicates that a single mutation confers recombination deficiency and the inability to lift immunity after UV irradiation. Possible relationships between recombination and the lifting of immunity are enumerated.

Katherine Brooks

Papers

Behavior of λ Bacteriophage in a Recombination Deficient Strain of Escherichia coli