scholarly journals Inducible Directed Evolution of Complex Phenotypes in Bacteria

2020 ◽  
Author(s):  
Ibrahim S. Al’Abri ◽  
Daniel J. Haller ◽  
Nathan Crook
Keyword(s):  
Escherichia Coli ◽  
Directed Evolution ◽  
Bacillus Licheniformis ◽  
Dna Sequences ◽  
Powerful Method ◽  
Temperate Bacteriophage ◽  
Gene Pathway ◽  
Complex Phenotypes ◽  
Large Plasmids ◽  
Lag Times

AbstractDirected evolution is a powerful method for engineering biology in the absence of detailed sequence-function relationships. To enable directed evolution of complex phenotypes encoded by multigene pathways, we require large library sizes for DNA sequences >5-10kb in length, elimination of genomic hitchhiker mutations, and decoupling of diversification and screening steps. To meet these challenges, we developed Inducible Directed Evolution (IDE), which uses a temperate bacteriophage to package large plasmids and transfer them to naive cells after intracellular mutagenesis. To demonstrate IDE, we evolved a 5-gene pathway from Bacillus licheniformis that accelerates tagatose catabolism in Escherichia coli, resulting in clones with 65% shorter lag times during growth on tagatose after only two rounds of evolution.

scholarly journals The DNA sequences encoding plsB and dgk loci of Escherichia coli.

1983 ◽  
Vol 258 (18) ◽  
pp. 10856-10861 ◽  
Author(s):  
V A Lightner ◽  
R M Bell ◽  
P Modrich
Keyword(s):  
Escherichia Coli ◽  
Dna Sequences

scholarly journals DNA sequences of the cysB regions of Salmonella typhimurium and Escherichia coli.

1987 ◽  
Vol 262 (13) ◽  
pp. 5999-6005 ◽  
Author(s):  
J. Ostrowski ◽  
G. Jagura-Burdzy ◽  
N.M. Kredich
Keyword(s):  
Escherichia Coli ◽  
Salmonella Typhimurium ◽  
Dna Sequences

scholarly journals Alignment of Escherichia coli K12 DNA sequences to a genomic restriction map

1990 ◽  
Vol 18 (2) ◽  
pp. 313-321 ◽  
Author(s):  
Kenneth E. Rudd ◽  
Webb Miller ◽  
James Ostell ◽  
Dennis A. Benson
Keyword(s):  
Escherichia Coli ◽  
Dna Sequences ◽  
Restriction Map ◽  
Escherichia Coli K12

scholarly journals Enhancement of the Efficiency of Secretion of Heterologous Lipase in Escherichia coli by Directed Evolution of the ABC Transporter System

2005 ◽  
Vol 71 (7) ◽  
pp. 3468-3474 ◽  
Author(s):  
Gyeong Tae Eom ◽  
Jae Kwang Song ◽  
Jung Hoon Ahn ◽  
Yeon Soo Seo ◽  
Joon Shick Rhee
Keyword(s):  
Escherichia Coli ◽  
Directed Evolution ◽  
Abc Transporter ◽  
Microtiter Plate ◽  
Single Amino Acid ◽  
Wild Type ◽  
E Coli ◽  
Single Amino Acid Change ◽  
Secretion Efficiency

ABSTRACT The ABC transporter (TliDEF) from Pseudomonas fluorescens SIK W1, which mediated the secretion of a thermostable lipase (TliA) into the extracellular space in Escherichia coli, was engineered using directed evolution (error-prone PCR) to improve its secretion efficiency. TliD mutants with increased secretion efficiency were identified by coexpressing the mutated tliD library with the wild-type tliA lipase in E. coli and by screening the library with a tributyrin-emulsified indicator plate assay and a microtiter plate-based assay. Four selected mutants from one round of error-prone PCR mutagenesis, T6, T8, T24, and T35, showed 3.2-, 2.6-, 2.9-, and 3.0-fold increases in the level of secretion of TliA lipase, respectively, but had almost the same level of expression of TliD in the membrane as the strain with the wild-type TliDEF transporter. These results indicated that the improved secretion of TliA lipase was mediated by the transporter mutations. Each mutant had a single amino acid change in the predicted cytoplasmic regions in the membrane domain of TliD, implying that the corresponding region of TliD was important for the improved and successful secretion of the target protein. We therefore concluded that the efficiency of secretion of a heterologous protein in E. coli can be enhanced by in vitro engineering of the ABC transporter.

scholarly journals Yeast DNA sequences initiating gene expression in Escherichia coli

2004 ◽  
Vol 159 (1) ◽  
pp. 19-28 ◽  
Author(s):  
Astrid Lewin ◽  
Thi Tuyen Tran ◽  
Daniela Jacob ◽  
Martin Mayer ◽  
Barbara Freytag ◽  
...  
Keyword(s):  
Gene Expression ◽  
Escherichia Coli ◽  
Dna Sequences ◽  
Expression In Escherichia Coli

scholarly journals Directed Evolution of Ionizing Radiation Resistance in Escherichia coli

2009 ◽  
Vol 191 (16) ◽  
pp. 5240-5252 ◽  
Author(s):  
Dennis R. Harris ◽  
Steve V. Pollock ◽  
Elizabeth A. Wood ◽  
Reece J. Goiffon ◽  
Audrey J. Klingele ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Ionizing Radiation ◽  
Directed Evolution ◽  
Radiation Resistance ◽  
Bacterial Species ◽  
Repair System ◽  
Multiple Alleles ◽  
Evolutionary Pathways ◽  
K 12 ◽  
Ionizing Radiation Resistance

ABSTRACT We have generated extreme ionizing radiation resistance in a relatively sensitive bacterial species, Escherichia coli, by directed evolution. Four populations of Escherichia coli K-12 were derived independently from strain MG1655, with each specifically adapted to survive exposure to high doses of ionizing radiation. D37 values for strains isolated from two of the populations approached that exhibited by Deinococcus radiodurans. Complete genomic sequencing was carried out on nine purified strains derived from these populations. Clear mutational patterns were observed that both pointed to key underlying mechanisms and guided further characterization of the strains. In these evolved populations, passive genomic protection is not in evidence. Instead, enhanced recombinational DNA repair makes a prominent but probably not exclusive contribution to genome reconstitution. Multiple genes, multiple alleles of some genes, multiple mechanisms, and multiple evolutionary pathways all play a role in the evolutionary acquisition of extreme radiation resistance. Several mutations in the recA gene and a deletion of the e14 prophage both demonstrably contribute to and partially explain the new phenotype. Mutations in additional components of the bacterial recombinational repair system and the replication restart primosome are also prominent, as are mutations in genes involved in cell division, protein turnover, and glutamate transport. At least some evolutionary pathways to extreme radiation resistance are constrained by the temporally ordered appearance of specific alleles.

scholarly journals Contributions of individual mechanisms to fluoroquinolone resistance in 36 Escherichia coli strains isolated from humans and animals.

1996 ◽  
Vol 40 (10) ◽  
pp. 2380-2386 ◽  
Author(s):  
M J Everett ◽  
Y F Jin ◽  
V Ricci ◽  
L J Piddock
Keyword(s):  
Escherichia Coli ◽  
Dna Sequences ◽  
Quinolone Resistance ◽  
Fluoroquinolone Resistance ◽  
Polymorphism Analysis ◽  
Single Mutation ◽  
Wild Type ◽  
Single Strand Conformational Polymorphism ◽  
E Coli ◽  
High Level

Twenty-eight human isolates of Escherichia coli from Argentina and Spain and eight veterinary isolates received from the Ministry of Agriculture Fisheries and Foods in the United Kingdom required 2 to > 128 micrograms of ciprofloxacin per ml for inhibition. Fragments of gyrA and parC encompassing the quinolone resistance-determining region were amplified by PCR, and the DNA sequences of the fragments were determined. All isolates contained a mutation in gyrA of a serine at position 83 (Ser83) to an Leu, and 26 isolates also contained a mutation of Asp87 to one of four amino acids: Asn (n = 14), Tyr (n = 6), Gly (n = 5), or His (n = 1). Twenty-four isolates contained a single mutation in parC, either a Ser80 to Ile (n = 17) or Arg (n = 2) or a Glu84 to Lys (n = 3). The role of a mutation in gyrB was investigated by introducing wild-type gyrB (pBP548) into all isolates; for three transformants MICs of ciprofloxacin were reduced; however, sequencing of PCR-derived fragments containing the gyrB quinolone resistance-determining region revealed no changes. The analogous region of parE was analyzed in 34 of 36 isolates by single-strand conformational polymorphism analysis and sequencing; however, no amino acid substitutions were discovered. The outer membrane protein and lipopolysaccharide profiles of all isolates were compared with those of reference strains, and the concentration of ciprofloxacin accumulated (with or without 100 microM carbony cyanide m-chlorophenylhydrazone [CCCP] was determined. Twenty-two isolates accumulated significantly lower concentrations of ciprofloxacin than the wild-type E. coli isolate; nine isolates accumulated less then half the concentration. The addition of CCCP increased the concentration of ciprofloxacin accumulated, and in all but one isolate the percent increase was greater than that in the control strains. The data indicate that high-level fluoroquinolone resistance in E. coli involves the acquisition of mutations at multiple loci.

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