Copy number variability of expression plasmids determined by cell sorting and Droplet Digital PCR

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Abstract

Background

Plasmids are widely used for molecular cloning or production of proteins in laboratory and industrial settings. Constant modification has brought forth countless plasmid vectors whose characteristics in terms of average plasmid copy number (PCN) and stability are rarely known. The crucial factor determining the PCN is the replication system; most replication systems in use today belong to a small number of different classes and are available through repositories like the Standard European Vector Architecture (SEVA).

Results

In this study, the PCN was determined in a set of seven SEVA-based expression plasmids only differing in the replication system. The average PCN for all constructs was determined by Droplet Digital PCR and ranged between 2 and 40 per chromosome in the host organism Escherichia coli. Furthermore, a plasmid-encoded EGFP reporter protein served as a means to assess variability in reporter gene expression on the single cell level. Only cells with one type of plasmid (RSF1010 replication system) showed a high degree of heterogeneity with a clear bimodal distribution of EGFP intensity while the others showed a normal distribution. The heterogeneous RSF1010-carrying cell population and one normally distributed population (ColE1 replication system) were further analyzed by sorting cells of sub-populations selected according to EGFP intensity. For both plasmids, low and highly fluorescent sub-populations showed a remarkable difference in PCN, ranging from 9.2 to 123.4 for ColE1 and from 0.5 to 11.8 for RSF1010, respectively.

Conclusions

The average PCN determined here for a set of standardized plasmids was generally at the lower end of previously reported ranges and not related to the degree of heterogeneity. Further characterization of a heterogeneous and a homogeneous population demonstrated considerable differences in the PCN of sub-populations. We therefore present direct molecular evidence that the average PCN does not represent the true number of plasmid molecules in individual cells.

Electronic supplementary material

The online version of this article (doi:10.1186/s12934-016-0610-8) contains supplementary material, which is available to authorized users.

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Most cited references 38

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Bistability, epigenetics, and bet-hedging in bacteria.

Jan-Willem Veening, Wiep Klaas Smits, Oscar P. Kuipers (2008)

Clonal populations of microbial cells often show a high degree of phenotypic variability under homogeneous conditions. Stochastic fluctuations in the cellular components that determine cellular states can cause two distinct subpopulations, a property called bistability. Phenotypic heterogeneity can be readily obtained by interlinking multiple gene regulatory pathways, effectively resulting in a genetic logic-AND gate. Although switching between states can occur within the cells' lifetime, cells can also pass their cellular state over to the next generation by a mechanism known as epigenetic inheritance and thus perpetuate the phenotypic state. Importantly, heterogeneous populations can demonstrate increased fitness compared with homogeneous populations. This suggests that microbial cells employ bet-hedging strategies to maximize survival. Here, we discuss the possible roles of interlinked bistable networks, epigenetic inheritance, and bet-hedging in bacteria.

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Replication of an origin-containing derivative of plasmid RK2 dependent on a plasmid function provided in trans.

D Figurski, D Helinski (1979)

pRK212.2, a derivative of the broad host range plasmid RK2, contains two EcoRI cleavage fragments, A and B, neither of which can replicate by itself in Escherichia coli. Fragment A (41.7 kilobases), but not fragment B (14.4 kilobases), can be cloned by insertion into the unrelated plasmids mini-F and ColE1. Fragment B contains the origin of replication and the ampicillin-resistance determinant of RK2. Transformation of E. coli cells containing the mini-F-fragment A hybrid plasmid with fragment B DNA results in the recircularization and replication of fragment B as a nonmobilizable plasmid (pRK2067) with the copy number and incompatibility properties of RK2. Fragment B cannot be cloned in the absence of fragment A because the latter fragment suppresses a function, specified by fragment B, that results in loss of host cell viability. A small segment (2.4 kilobases) of fragment B that contains the RK2 origin of replication but no longer affects host cell growth in the absence of fragment A had been cloned previously by insertion into a ColE1 plasmid. This hybrid plasmid, designated pRK256, will replicate in E. coli polA mutants only when a fragment A-bearing helper plasmid is present. These results demonstrate that the potentially lethal function specified by fragment B of RK2 is not necessary for replication and that at least one trans-acting function is directly involved in RK2 replication.

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Transposon vectors containing non-antibiotic resistance selection markers for cloning and stable chromosomal insertion of foreign genes in gram-negative bacteria.

M. Herrero, V de Lorenzo, K Timmis (1990)

A simple procedure for cloning and stable insertion of foreign genes into the chromosomes of gram-negative eubacteria was developed by combining in two sets of plasmids (i) the transposition features of Tn10 and Tn5; (ii) the resistances to the herbicide bialaphos, to mercuric salts and organomercurial compounds, and to arsenite, and (iii) the suicide delivery properties of the R6K-based plasmid pGP704. The resulting constructions contained unique NotI or SfiI sites internal to either the Tn10 or the Tn5 inverted repeats. These sites were readily used for cloning DNA fragments with the help of two additional specialized cloning plasmids, pUC18Not and pUC18Sfi. The newly derived constructions could be maintained only in donor host strains that produce the R6K-specified pi protein, which is an essential replication protein for R6K and plasmids derived therefrom. Donor plasmids containing hybrid transposons were transformed into a specialized lambda pir lysogenic Escherichia coli strain with a chromosomally integrated RP4 that provided broad-host-range conjugal transfer functions. Delivery of the donor plasmids into selected host bacteria was accomplished through mating with the target strain. Transposition of the hybrid transposon from the delivered suicide plasmid to a replicon in the target cell was mediated by the cognate transposase encoded on the plasmid at a site external to the transposon. Since the transposase function was not maintained in target cells, such cells were not immune to further transposition rounds. Multiple insertions in the same strain are therefore only limited by the availability of distinct selection markers. The utility of the system was demonstrated with a kanamycin resistance gene as a model foreign insert into Pseudomonas putida and a melanin gene from Streptomyces antibioticus into Klebsiella pneumoniae. Because of the modular nature of the functional parts of the cloning vectors, they can be easily modified and further selection markers can be incorporated. The cloning system described here will be particularly useful for the construction of hybrid bacteria that stably maintain inserted genes, perhaps in competitive situations (e.g., in open systems and natural environments), and that do not carry antibiotic resistance markers characteristic of most available cloning vectors (as is currently required of live bacterial vaccines).

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Author and article information

Contributors

Michael Jahn:

ORCID: http://orcid.org/0000-0002-3913-153X

michael.jahn@scilifelab.se

Carsten Vorpahl: v_carsten@web.de

Thomas Hübschmann: thomas.huebschmann@ufz.de

Hauke Harms: hauke.harms@ufz.de

Susann Müller: +49 341 235 1318 , susann.mueller@ufz.de

Journal

Journal ID (nlm-ta): Microb Cell Fact

Journal ID (iso-abbrev): Microb. Cell Fact

Title: Microbial Cell Factories

Publisher: BioMed Central (London )

ISSN (Electronic): 1475-2859

Publication date (Electronic): 19 December 2016

Publication date PMC-release: 19 December 2016

Publication date Collection: 2016

Volume: 15

Electronic Location Identifier: 211

Affiliations

[1 ]Helmholtz-Centre for Environmental Research-UFZ, Permoserstraße 15, 04318 Leipzig, Germany

[2 ]School of Biotechnology, Science for Life Laboratory, KTH-Royal Institute of Technology, Stockholm, Sweden

Author information

Michael Jahn http://orcid.org/0000-0002-3913-153X

Article

Publisher ID: 610

DOI: 10.1186/s12934-016-0610-8

PMC ID: 5168713

PubMed ID: 27993152

SO-VID: b5e24564-a1b2-426f-bcc6-56db874d9036

License:

Open AccessThis article is distributed under the terms of the Creative Commons Attribution 4.0 International License ( http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver ( http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.

History

Date received : 24 September 2016

Date accepted : 2 December 2016

Funding

Funded by: FundRef http://dx.doi.org/10.13039/501100006298, Sächsische Aufbaubank;

Award ID: 100074351

Award Recipient : Michael Jahn

Funded by: FundRef http://dx.doi.org/10.13039/501100004965, Sixth Framework Programme;

Award ID: 100074351

Award Recipient : Michael Jahn

Custom metadata

ScienceOpen disciplines: Biotechnology

Keywords: escherichia coli,seva,replication system,origin of replication,plasmid copy number,egfp,population heterogeneity,variability,cell sorting,sub-population

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ScienceOpen disciplines: Biotechnology

Keywords: escherichia coli, seva, replication system, origin of replication, plasmid copy number, egfp, population heterogeneity, variability, cell sorting, sub-population

Copy number variability of expression plasmids determined by cell sorting and Droplet Digital PCR

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Most cited references 38

Bistability, epigenetics, and bet-hedging in bacteria.

Replication of an origin-containing derivative of plasmid RK2 dependent on a plasmid function provided in trans.

Transposon vectors containing non-antibiotic resistance selection markers for cloning and stable chromosomal insertion of foreign genes in gram-negative bacteria.

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