Directed evolution and identification of control regions of ColE1 plasmid replication origins using only nucleotide deletions.
Genes can be mutated by altering DNA content (base changes) or DNA length (insertions or deletions). Most in vitro directed evolution processes utilize nucleotide content changes to produce DNA libraries. We tested whether gain of function mutations could be identified using a mutagenic process that produced only nucleotide deletions. Short nucleotide stretches were deleted in a plasmid encoding lacZ, and screened for increased beta-galactosidase activity. Several mutations were found in the origin of replication that quantitatively and qualitatively altered plasmid behavior in vivo. Some mutations allowed co-residence of ColE1 plasmids in Escherichia coli, and implicate hairpin structures II and III of the ColE1 RNA primer as determinants of plasmid compatibility. Thus, useful and unexpected mutations can be found from libraries containing only deletions.
Kim
Dewey
D
IntegriGen, Inc., 42 Digital Dr. Bldg. 6, Novato, CA 94949, USA.
Rhee
Yoon
Y
Rhodes
Denise
D
Sharma
Vikram
V
Sorenson
Olav
O
Greener
Alan
A
Smider
Vaughn
V
D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000235:genetics; D001483:Base Sequence; D004269:DNA, Bacterial / Q000235:genetics; D019020:Directed Molecular Evolution; D029968:Escherichia coli Proteins / Q000235:genetics; D005798:Genes, Bacterial; D007763:Lac Operon; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D010641:Phenotype; D010957:Plasmids / Q000235:genetics*; D012329:RNA, Bacterial / Q000737:chemistry / Q000235:genetics; D018741:Replication Origin; D017384:Sequence Deletion; D012689:Sequence Homology, Nucleic Acid
D001426:Bacterial Proteins; D004269:DNA, Bacterial; D029968:Escherichia coli Proteins; D012329:RNA, Bacterial; C042833:ROM protein, Bacteria