Comparative Single-Cell Analysis of Different E. coli Expression Systems during Microfluidic Cultivation

Binder D, Probst C, Grünberger A, Hilgers F, Loeschcke A, Jaeger K-E, Kohlheyer D, Drepper T (2016)
PLoS one 11(8): e0160711.

Zeitschriftenaufsatz | Veröffentlicht | Englisch
 
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Binder, Dennis; Probst, Christopher; Grünberger, AlexanderUniBi; Hilgers, Fabienne; Loeschcke, Anita; Jaeger, Karl-Erich; Kohlheyer, Dietrich; Drepper, Thomas
Abstract / Bemerkung
Recombinant protein production is mostly realized with large-scale cultivations and monitored at the level of the entire population. Detailed knowledge of cell-to-cell variations with respect to cellular growth and product formation is limited, even though phenotypic heterogeneity may distinctly hamper overall production yields, especially for toxic or difficult-to-express proteins. Unraveling phenotypic heterogeneity is thus a key aspect in understanding and optimizing recombinant protein production in biotechnology and synthetic biology. Here, microfluidic single-cell analysis serves as the method of choice to investigate and unmask population heterogeneities in a dynamic and spatiotemporal fashion. In this study, we report on comparative microfluidic single-cell analyses of commonly used E. coli expression systems to uncover system-inherent specifications in the synthetic M9CA growth medium. To this end, the PT7lac/LacI, the PBAD/AraC and the Pm/XylS system were systematically analyzed in order to gain detailed insights into variations of growth behavior and expression phenotypes and thus to uncover individual strengths and deficiencies at the single-cell level. Specifically, we evaluated the impact of different system-specific inducers, inducer concentrations as well as genetic modifications that affect inducer-uptake and regulation of target gene expression on responsiveness and phenotypic heterogeneity. Interestingly, the most frequently applied expression system based on E. coli strain BL21(DE3) clearly fell behind with respect to expression homogeneity and robustness of growth. Moreover, both the choice of inducer and the presence of inducer uptake systems proved crucial for phenotypic heterogeneity. Conclusively, microfluidic evaluation of different inducible E. coli expression systems and setups identified the modified lacY-deficient PT7lac/LacI as well as the Pm/XylS system with conventional m-toluic acid induction as key players for precise and robust triggering of bacterial gene expression in E. coli in a homogeneous fashion.
Erscheinungsjahr
2016
Zeitschriftentitel
PLoS one
Band
11
Ausgabe
8
Art.-Nr.
e0160711
ISBN
1932-6203
ISSN
1932-6203
Page URI
https://pub.uni-bielefeld.de/record/2912514

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Binder D, Probst C, Grünberger A, et al. Comparative Single-Cell Analysis of Different E. coli Expression Systems during Microfluidic Cultivation. PLoS one. 2016;11(8): e0160711.
Binder, D., Probst, C., Grünberger, A., Hilgers, F., Loeschcke, A., Jaeger, K. - E., Kohlheyer, D., et al. (2016). Comparative Single-Cell Analysis of Different E. coli Expression Systems during Microfluidic Cultivation. PLoS one, 11(8), e0160711. doi:10.1371/journal.pone.0160711
Binder, Dennis, Probst, Christopher, Grünberger, Alexander, Hilgers, Fabienne, Loeschcke, Anita, Jaeger, Karl-Erich, Kohlheyer, Dietrich, and Drepper, Thomas. 2016. “Comparative Single-Cell Analysis of Different E. coli Expression Systems during Microfluidic Cultivation”. PLoS one 11 (8): e0160711.
Binder, D., Probst, C., Grünberger, A., Hilgers, F., Loeschcke, A., Jaeger, K. - E., Kohlheyer, D., and Drepper, T. (2016). Comparative Single-Cell Analysis of Different E. coli Expression Systems during Microfluidic Cultivation. PLoS one 11:e0160711.
Binder, D., et al., 2016. Comparative Single-Cell Analysis of Different E. coli Expression Systems during Microfluidic Cultivation. PLoS one, 11(8): e0160711.
D. Binder, et al., “Comparative Single-Cell Analysis of Different E. coli Expression Systems during Microfluidic Cultivation”, PLoS one, vol. 11, 2016, : e0160711.
Binder, D., Probst, C., Grünberger, A., Hilgers, F., Loeschcke, A., Jaeger, K.-E., Kohlheyer, D., Drepper, T.: Comparative Single-Cell Analysis of Different E. coli Expression Systems during Microfluidic Cultivation. PLoS one. 11, : e0160711 (2016).
Binder, Dennis, Probst, Christopher, Grünberger, Alexander, Hilgers, Fabienne, Loeschcke, Anita, Jaeger, Karl-Erich, Kohlheyer, Dietrich, and Drepper, Thomas. “Comparative Single-Cell Analysis of Different E. coli Expression Systems during Microfluidic Cultivation”. PLoS one 11.8 (2016): e0160711.
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7 Zitationen in Europe PMC

Daten bereitgestellt von Europe PubMed Central.

Growth-dependent recombinant product formation kinetics can be reproduced through engineering of glucose transport and is prone to phenotypic heterogeneity.
Fragoso-Jiménez JC, Baert J, Nguyen TM, Liu W, Sassi H, Goormaghtigh F, Van Melderen L, Gaytán P, Hernández-Chávez G, Martinez A, Delvigne F, Gosset G., Microb Cell Fact 18(1), 2019
PMID: 30710996
Plasmids for Independently Tunable, Low-Noise Expression of Two Genes.
Silva JPN, Lopes SV, Grilo DJ, Hensel Z., mSphere 4(3), 2019
PMID: 31142623
Homogenizing bacterial cell factories: Analysis and engineering of phenotypic heterogeneity.
Binder D, Drepper T, Jaeger KE, Delvigne F, Wiechert W, Kohlheyer D, Grünberger A., Metab Eng 42(), 2017
PMID: 28645641

57 References

Daten bereitgestellt von Europe PubMed Central.

Bistability, epigenetics, and bet-hedging in bacteria.
Veening JW, Smits WK, Kuipers OP., Annu. Rev. Microbiol. 62(), 2008
PMID: 18537474
Functional roles for noise in genetic circuits
AUTHOR UNKNOWN, 2010
Synthetic biology: advancing the design of diverse genetic systems.
Wang YH, Wei KY, Smolke CD., Annu Rev Chem Biomol Eng 4(), 2013
PMID: 23413816
Designing and engineering evolutionary robust genetic circuits.
Sleight SC, Bartley BA, Lieviant JA, Sauro HM., J Biol Eng 4(), 2010
PMID: 21040586
A comparative analysis of the properties of regulated promoter systems commonly used for recombinant gene expression in Escherichia coli.
Balzer S, Kucharova V, Megerle J, Lale R, Brautaset T, Valla S., Microb. Cell Fact. 12(), 2013
PMID: 23506076
Positively regulated bacterial expression systems.
Brautaset T, Lale R, Valla S., Microb Biotechnol 2(1), 2008
PMID: 21261879
Recombinant protein expression in Escherichia coli: advances and challenges.
Rosano GL, Ceccarelli EA., Front Microbiol 5(), 2014
PMID: 24860555

AUTHOR UNKNOWN, 2011
Protein production and purification.
Structural Genomics Consortium; China Structural Genomics Consortium; Northeast Structural Genomics Consortium, Graslund S, Nordlund P, Weigelt J, Hallberg BM, Bray J, Gileadi O, Knapp S, Oppermann U, Arrowsmith C, Hui R, Ming J, dhe-Paganon S, Park HW, Savchenko A, Yee A, Edwards A, Vincentelli R, Cambillau C, Kim R, Kim SH, Rao Z, Shi Y, Terwilliger TC, Kim CY, Hung LW, Waldo GS, Peleg Y, Albeck S, Unger T, Dym O, Prilusky J, Sussman JL, Stevens RC, Lesley SA, Wilson IA, Joachimiak A, Collart F, Dementieva I, Donnelly MI, Eschenfeldt WH, Kim Y, Stols L, Wu R, Zhou M, Burley SK, Emtage JS, Sauder JM, Thompson D, Bain K, Luz J, Gheyi T, Zhang F, Atwell S, Almo SC, Bonanno JB, Fiser A, Swaminathan S, Studier FW, Chance MR, Sali A, Acton TB, Xiao R, Zhao L, Ma LC, Hunt JF, Tong L, Cunningham K, Inouye M, Anderson S, Janjua H, Shastry R, Ho CK, Wang D, Wang H, Jiang M, Montelione GT, Stuart DI, Owens RJ, Daenke S, Schutz A, Heinemann U, Yokoyama S, Bussow K, Gunsalus KC., Nat. Methods 5(2), 2008
PMID: 18235434
Tuning Escherichia coli for membrane protein overexpression.
Wagner S, Klepsch MM, Schlegel S, Appel A, Draheim R, Tarry M, Hogbom M, van Wijk KJ, Slotboom DJ, Persson JO, de Gier JW., Proc. Natl. Acad. Sci. U.S.A. 105(38), 2008
PMID: 18796603
Light-responsive control of bacterial gene expression: precise triggering of the lac promoter activity using photocaged IPTG.
Binder D, Grunberger A, Loeschcke A, Probst C, Bier C, Pietruszka J, Wiechert W, Kohlheyer D, Jaeger KE, Drepper T., Integr Biol (Camb) 6(8), 2014
PMID: 24894989
Bacteriophage T7 RNA polymerase travels far ahead of ribosomes in vivo.
Iost I, Guillerez J, Dreyfus M., J. Bacteriol. 174(2), 1992
PMID: 1729251
Crystal structure of the lactose operon repressor and its complexes with DNA and inducer.
Lewis M, Chang G, Horton NC, Kercher MA, Pace HC, Schumacher MA, Brennan RG, Lu P., Science 271(5253), 1996
PMID: 8638105
Structural analysis of lac repressor bound to allosteric effectors.
Daber R, Stayrook S, Rosenberg A, Lewis M., J. Mol. Biol. 370(4), 2007
PMID: 17543986
Genetic regulatory mechanisms in the synthesis of proteins.
JACOB F, MONOD J., J. Mol. Biol. 3(), 1961
PMID: 13718526
Multistability in the lactose utilization network of Escherichia coli.
Ozbudak EM, Thattai M, Lim HN, Shraiman BI, Van Oudenaarden A., Nature 427(6976), 2004
PMID: 14973486
Lessons from lactose permease.
Guan L, Kaback HR., Annu Rev Biophys Biomol Struct 35(), 2006
PMID: 16689628
Transport of galactose, glucose and their molecular analogues by Escherichia coli K12.
Henderson PJ, Giddens RA, Jones-Mortimer MC., Biochem. J. 162(2), 1977
PMID: 15558
Evidencing the role of lactose permease in IPTG uptake by Escherichia coli in fed-batch high cell density cultures.
Fernandez-Castane A, Vine CE, Caminal G, Lopez-Santin J., J. Biotechnol. 157(3), 2011
PMID: 22202176
Tight regulation, modulation, and high-level expression by vectors containing the arabinose PBAD promoter.
Guzman LM, Belin D, Carson MJ, Beckwith J., J. Bacteriol. 177(14), 1995
PMID: 7608087
Single cell kinetics of phenotypic switching in the arabinose utilization system of E. coli.
Fritz G, Megerle JA, Westermayer SA, Brick D, Heermann R, Jung K, Radler JO, Gerland U., PLoS ONE 9(2), 2014
PMID: 24586851
Random mutagenesis of the PM promoter as a powerful strategy for improvement of recombinant-gene expression.
Bakke I, Berg L, Aune TE, Brautaset T, Sletta H, Tondervik A, Valla S., Appl. Environ. Microbiol. 75(7), 2009
PMID: 19201973
A propionate-inducible expression system for enteric bacteria.
Lee SK, Keasling JD., Appl. Environ. Microbiol. 71(11), 2005
PMID: 16269719
A disposable picolitre bioreactor for cultivation and investigation of industrially relevant bacteria on the single cell level.
Grunberger A, Paczia N, Probst C, Schendzielorz G, Eggeling L, Noack S, Wiechert W, Kohlheyer D., Lab Chip 12(11), 2012
PMID: 22511122
Microfluidic picoliter bioreactor for microbial single-cell analysis: fabrication, system setup, and operation
AUTHOR UNKNOWN, 2013
Spatiotemporal microbial single-cell analysis using a high-throughput microfluidics cultivation platform.
Grunberger A, Probst C, Helfrich S, Nanda A, Stute B, Wiechert W, von Lieres E, Noh K, Frunzke J, Kohlheyer D., Cytometry A 87(12), 2015
PMID: 26348020
NIH Image to ImageJ: 25 years of image analysis.
Schneider CA, Rasband WS, Eliceiri KW., Nat. Methods 9(7), 2012
PMID: 22930834
Beyond growth rate 0.6: Corynebacterium glutamicum cultivated in highly diluted environments.
Grunberger A, van Ooyen J, Paczia N, Rohe P, Schiendzielorz G, Eggeling L, Wiechert W, Kohlheyer D, Noack S., Biotechnol. Bioeng. 110(1), 2012
PMID: 22890752
Technical bias of microcultivation environments on single-cell physiology.
Dusny C, Grunberger A, Probst C, Wiechert W, Kohlheyer D, Schmid A., Lab Chip 15(8), 2015
PMID: 25710324

AUTHOR UNKNOWN, 1989
A rapid and efficient method for site-directed mutagenesis using one-step overlap extension PCR.
Urban A, Neukirchen S, Jaeger KE., Nucleic Acids Res. 25(11), 1997
PMID: 9153325
Single-cell microfluidics: opportunity for bioprocess development.
Grunberger A, Wiechert W, Kohlheyer D., Curr. Opin. Biotechnol. 29(), 2014
PMID: 24642389
Expression of highly toxic genes in E. coli: special strategies and genetic tools.
Saida F, Uzan M, Odaert B, Bontems F., Curr. Protein Pept. Sci. 7(1), 2006
PMID: 16472168
Phenotyping the quality of complex medium components by simple online-monitored shake flask experiments.
Diederichs S, Korona A, Staaden A, Kroutil W, Honda K, Ohtake H, Buchs J., Microb. Cell Fact. 13(), 2014
PMID: 25376163
A novel T7 RNA polymerase dependent expression system for high-level protein production in the phototrophic bacterium Rhodobacter capsulatus.
Katzke N, Arvani S, Bergmann R, Circolone F, Markert A, Svensson V, Jaeger KE, Heck A, Drepper T., Protein Expr. Purif. 69(2), 2009
PMID: 19706327
Photocaged Arabinose: A Novel Optogenetic Switch for Rapid and Gradual Control of Microbial Gene Expression.
Binder D, Bier C, Grunberger A, Drobietz D, Hage-Hulsmann J, Wandrey G, Buchs J, Kohlheyer D, Loeschcke A, Wiechert W, Jaeger KE, Pietruszka J, Drepper T., Chembiochem 17(4), 2016
PMID: 26677142
High-throughput gene expression analysis at the level of single proteins using a microfluidic turbidostat and automated cell tracking.
Ullman G, Wallden M, Marklund EG, Mahmutovic A, Razinkov I, Elf J., Philos. Trans. R. Soc. Lond., B, Biol. Sci. 368(1611), 2012
PMID: 23267179
Streaming instability in growing cell populations.
Mather W, Mondragon-Palomino O, Danino T, Hasty J, Tsimring LS., Phys. Rev. Lett. 104(20), 2010
PMID: 20867071
Directed evolution of AraC for improved compatibility of arabinose- and lactose-inducible promoters.
Lee SK, Chou HH, Pfleger BF, Newman JD, Yoshikuni Y, Keasling JD., Appl. Environ. Microbiol. 73(18), 2007
PMID: 17644634
Charting microbial phenotypes in multiplex nanoliter batch bioreactors.
Dai J, Yoon SH, Sim HY, Yang YS, Oh TK, Kim JF, Hong JW., Anal. Chem. 85(12), 2013
PMID: 23581968
Pre-dispositions and epigenetic inheritance in the Escherichia coli lactose operon bistable switch
AUTHOR UNKNOWN, 2010
Regulatable arabinose-inducible gene expression system with consistent control in all cells of a culture.
Khlebnikov A, Risa O, Skaug T, Carrier TA, Keasling JD., J. Bacteriol. 182(24), 2000
PMID: 11092865
Homogeneous expression of the P(BAD) promoter in Escherichia coli by constitutive expression of the low-affinity high-capacity AraE transporter.
Khlebnikov A, Datsenko KA, Skaug T, Wanner BL, Keasling JD., Microbiology (Reading, Engl.) 147(Pt 12), 2001
PMID: 11739756
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