T7 Expression Systems for Inducible Production of Proteins from Cloned Genes in E. coli.

Inducible T7 expression systems are capable of producing a wide range of proteins in E. coli. Improvements over common practice include: (1) preventing unintended induction by establishing and maintaining expression strains in non-inducing growth media composed entirely of purified components instead of complex growth media that may variably induce target proteins on approach to saturation; and (2) expressing many target proteins in parallel by convenient and productive auto-induction in BL21(DE3) and other suitable hosts, instead of IPTG induction.

Recombinant transfer in the basic genome of Escherichia coli.

An approximation to the ∼4-Mbp basic genome shared by 32 strains of Escherichia coli representing six evolutionary groups has been derived and analyzed computationally. A multiple alignment of the 32 complete genome sequences was filtered to remove mobile elements and identify the most reliable ∼90% of the aligned length of each of the resulting 496 basic-genome pairs. Patterns of single base-pair mutations (SNPs) in aligned pairs distinguish clonally inherited regions from regions where either genome has acquired DNA fragments from diverged genomes by homologous recombination since their last common ancestor.

Stable expression clones and auto-induction for protein production in E. coli.

Inducible production of proteins from cloned genes in E. coli is widely used, economical, and effective. However, common practices can result in unintended induction, inadvertently generating cultures that give poor or variable yields in protein production.

Genome sequences of Escherichia coli B strains REL606 and BL21(DE3).

Escherichia coli K-12 and B have been the subjects of classical experiments from which much of our understanding of molecular genetics has emerged. We present here complete genome sequences of two E. coli B strains, REL606, used in a long-term evolution experiment, and BL21(DE3), widely used to express recombinant proteins.

Understanding the differences between genome sequences of Escherichia coli B strains REL606 and BL21(DE3) and comparison of the E. coli B and K-12 genomes.

Each difference between the genome sequences of Escherichia coli B strains REL606 and BL21(DE3) can be interpreted in light of known laboratory manipulations plus a gene conversion between ribosomal RNA operons. Two treatments with 1-methyl-3-nitro-1-nitrosoguanidine in the REL606 lineage produced at least 93 single-base-pair mutations ( approximately 90% GC-to-AT transitions) and 3 single-base-pair GC deletions. Two UV treatments in the BL21(DE3) lineage produced only 4 single-base-pair mutations but 16 large deletions.