Abstract
The availability of a robust and reliable continuous culture apparatus that eliminates wall growth problems would lead to many applications in the microbial field, including allowing genetically engineered strains to recover high fitness, improving biodegradation strains, and predicting likely antibiotic resistance mechanisms. We describe the design and implementation of a novel automated continuous culture machine that can be used both in time-dependent mode (similar to a chemostat) and turbidostat modes, in which wall growth is circumvented through the use of a long, variably divisible tube of growth medium. This tube can be restricted with clamps to create a mobile growth chamber region in which static portions of the tube and the associated medium are replaced together at equal rates. To functionally test the device as a tool for re-adaptation of engineered strains, we evolved a strain carrying a highly deleterious deletion of Elongation Factor P, a gene involved in translation. In 200 generations over 2 weeks of dilution cycles, the evolved strain improved in generation time by a factor of three, with no contaminations and easy manipulation.
This is a preview of subscription content, log in via an institution to check access.
Similar content being viewed by others
References
Aoki H, Dekany K, Adams SL, Ganoza MC (1997) The gene encoding the elongation factor P protein is essential for viability and is required for protein synthesis. J Biol Chem 272:32254–32259
Bonomo J, Warnecke T, Hume P, Marizcurrena A, Gill RT (2006) A comparative study of metabolic engineering anti-metabolite tolerance in Escherichia coli. Metab Eng 8:227–239
Bryson V, Szybakski W (1952) Microbial selection. Science 116:45–51
de Crécy E (2005) Continuous culture apparatus with mobile vessel allowing selection of fitter cell variants. WO/2005/083052
de Crécy-Lagard VA, Bellalou J, Mutzel R, Marlière P (2001) Long term adaptation of a microbial population to a permanent metabolic constraint: overcoming thymineless death by experimental evolution of Escherichia coli. BMC Biotechnol 1:10
Dykhuizen DE (1993) Chemostats used for studying natural selection and adaptive evolution. Methods Enzymol 224:613–631
Dykhuizen DE, Hartl DL (1983) Selection in chemostats. Microbiol Rev 47:150–168
Fong SS, Palsson BO (2004) Metabolic gene-deletion strains of Escherichia coli evolve to computationally predicted growth phenotypes. Nat Genet 36:1056–1058
Fong SS, Burgard AP, Herring CD, Knight EM, Blattner FR, Maranas CD, Palsson BO (2005) In silico design and adaptive evolution of Escherichia coli for production of lactic acid. Biotechnol Bioeng 91:643–648
Fong SS, Nanchen A, Palsson BO, Sauer U (2006) Latent pathway activation and increased pathway capacity enable Escherichia coli adaptation to loss of key metabolic enzymes. J Biol Chem 281:8024–8033
Ganoza MC, Aoki H (2000) Peptide bond synthesis: function of the efp gene product. Biol Chem 381:553–559
Helling RB, Kinney T, Adams J (1981) The maintenance of plasmid-containing organisms in populations of Escherichia coli. J Gen Microbiol 123:129–141
Larsen DH, Dimmick RL (1964) Attachment and growth of bacteria on surfaces of continuous culture vessels. J Bacteriol 88:1380–1387
Lenski RE (1993) Evaluating the fate of genetically modified microorganisms in the environment: are they inherently less fit? Experientia 49:201–209
Lenski RE, Travisano M (1994) Dynamics of adaptation and diversification: a 10,000-generation experiment with bacterial populations. Proc Natl Acad Sci USA 91:6808–6814
Metzgar D, Bacher JM, Pezo V, Reader J, Döring V, Schimmel P, Marlière P, de Crécy-Lagard V (2004) Acinetobacter sp. ADP1: an ideal model organism for genetic analysis and genome engineering. Nucleic Acids Res 32:5780–5790
Meynial-Salles I, Forchhammer N, Croux C, Girbal L, Soucaille P (2007) Evolution of a Saccharomyces cerevisiae metabolic pathway in Escherichia coli. Metab Eng 9:152–159
Monod J (1950) La technique de culture continue. Théorie et applications. Ann Inst Pasteur 19:390–410
Mutzel R, Mazel D, Marlière P (2003) Method for obtaining cells with new properties WO 03/004656
Notley-McRobb L, Ferenci T (1999) The generation of multiple co-existing mal-regulatory mutations through polygenic evolution in glucose-limited populations of Escherichia coli. Environ Microbiol 1:45–52
Novick A, Szilard L (1950a) Description of the chemostat. Science 112:715–716
Novick A, Szilard L (1950b) Experiments with the chemostat on spontaneous mutations of bacteria. Proc Natl Acad Sci USA 36:708–719
Spratt BG (1994) Resistance to antibiotics mediated by target alterations. Science 264:388–393
Swaney S, McCroskey M, Shinabarger D, Wang Z, Turner BA, Parker CN (2006) Characterization of a high-throughput screening assay for inhibitors of elongation factor p and ribosomal peptidyl transferase activity. J Biomol Screen 11:736–742
Vidal O, Longin R, Prigent-Combaret C, Dorel C, Hooreman M, Lejeune P (1998) Isolation of an Escherichia coli K-12 mutant strain able to form biofilms on inert surfaces: involvement of a new ompR allele that increases curli expression. J Bacteriol 180:2442–2449
Wahl LM, Gerrish PJ, Saika-Voivod I (2002) Evaluating the impact of population bottlenecks in experimental evolution. Genetics 162:961
Zhou S, Yomano LP, Shanmugam KT, Ingram LO (2005) Fermentation of 10% (w/v) sugar to D: (−)-lactate by engineered Escherichia coli B. Biotech Lett 27:1891–1896
Acknowledgements
We thank Phillippe Marlière for inspiration, Paul Schimmel for encouragements in the initial stages of the project, and Daniel Dykhuizen for critical reading of the manuscript. V d C-L work was funded in part by grant MCB-0128901 from the National Science Foundation.
Author information
Authors and Affiliations
Corresponding author
Rights and permissions
About this article
Cite this article
de Crécy, E., Metzgar, D., Allen, C. et al. Development of a novel continuous culture device for experimental evolution of bacterial populations. Appl Microbiol Biotechnol 77, 489–496 (2007). https://doi.org/10.1007/s00253-007-1168-5
Received:
Revised:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s00253-007-1168-5