Abstract
BackgroundTo maintain populations of microbial cells under controlled conditions of growth and environment for an indefinite duration is a prerequisite for experimentally evolving natural isolates of wild-type species or recombinant strains. This goal is beyond the scope of current continuous culture apparatus because these devices positively select mutants that evade dilution, primarily through attachment to vessel surfaces, resulting in persistent sub-populations of uncontrollable size and growth rate.ResultsTo overcome this drawback, a device with two growth chambers periodically undergoing transient phases of sterilization was designed. The robustness of this device was assessed by propagating an E. coli strain under permanent thymine starvation for over 880 days, i.e. metabolic conditions notoriously known to lead to cell death and clogging of cultivation vessels. Ten thousand generations were required to obtain a descendant lineage that could resist thymine starvation and had recovered wild-type growth rate.ConclusionsThis approach provides a technological framework for the diversification and improvement of microbial strains by long-term adaptation to inescapable metabolic constraints. An E. coli strain that is totally resistant to thymineless death was selected.
Highlights
Introduction to research with continuous culturesEdited by Prentice-Hall; 1970 2
Experimental evolution of microorganisms is a field of vast potential for both fundamental and industrial purposes
Genetic engineering masters the modular assembly of genes and their products, but it is by selecting for the enhancement of the overall fitness of recombinant organisms that we can most readily improve the functional integration of such assemblies
Summary
Introduction to research with continuous culturesEdited by Prentice-Hall; 1970 2. To maintain populations of microbial cells under controlled conditions of growth and environment for an indefinite duration is a prerequisite for experimentally evolving natural isolates of wild-type species or recombinant strains This goal is beyond the scope of current continuous culture apparatus because these devices positively select mutants that evade dilution, primarily through attachment to vessel surfaces, resulting in persistent sub-populations of uncontrollable size and growth rate. The long-term proliferation of microorganisms is covered by a body of knowledge known as continuous culture [1,2], technically and theoretically established around the equivalent Monod's bactogene [3] and Novick's and Szilard's chemostat [4] These devices operate by renewing a liquid culture of constant volume with nutrient medium inflow, such that microbes must counteract dilution by growing at least at an equal rate [1,2]. Mutants proliferating at higher growth rates are selected during prolonged operation of these devices [1,2]
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