Rapid optimization of gene dosage in E. coli using DIAL strains

Kittleson, Joshua T; Cheung, Sherine F.; Anderson, Janet S.

doi:10.1186/1754-1611-5-10

Cited by 57 publications

(68 citation statements)

References 33 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Importantly, there was also no relationship between average PCN and the degree of heterogeneity in terms of EGFP expression. This is in contrast to results obtained by Kittleson and colleagues [8] where a higher average PCN correlated with lower variability. However, we further characterized the variability of PCN on the sub-population level for two E. coli strains, one carrying a plasmid with the low-copy RSF1010 origin showing a bimodal distribution of EGFP fluorescence, and one with the high-copy ColE1 replication system having a unimodal distribution (mean PCN of 5.2 and 32, respectively).…”

Section: Resultscontrasting

confidence: 99%

See 1 more Smart Citation

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

et al. 2016

View full text Add to dashboard Cite

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

show abstract

Section: Resultscontrasting

confidence: 99%

“…As plasmids follow a discrete distribution, a low mean PCN may also lead to higher cell to cell heterogeneity regarding PCN and gene expression, as e.g. shown by Kittleson and colleagues using a library of 20 rep mutants [8]. …”

Section: Introductionmentioning

confidence: 99%

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

et al. 2016

View full text Add to dashboard Cite

show abstract

“…Different origins of replication can generate complex and poorly understood effects on expression, for example, by changing localization and supercoiling. This can be minimized by using plasmid systems where the copy number can be controlled without changing the origin 155 .…”

Section: Selecting Parts To Tune the Circuit Responsementioning

confidence: 99%

Principles of genetic circuit design

2014

View full text Add to dashboard Cite

Cells are able to navigate environments, communicate, and build complex patterns by initiating gene expression in response to specific signals. Engineers need to harness this capability to program cells to perform tasks or build chemicals and materials that match the complexity seen in nature. This review describes new tools that aid the construction of genetic circuits. We show how circuit dynamics can be influenced by the choice of regulators and changed with expression “tuning knobs.” We collate the failure modes encountered when assembling circuits, quantify their impact on performance, and review mitigation efforts. Finally, we discuss the constraints that arise from operating within a living cell. Collectively, better tools, well-characterized parts, and a comprehensive understanding of how to compose circuits are leading to a breakthrough in the ability to program living cells for advanced applications, from living therapeutics to the atomic manufacturing of functional materials.

show abstract

“…They can be manipulated quickly and easily, and a variety of replicons for use in Escherichia coli have become available (3), allowing, e.g., different expression levels by using plasmids with different copy numbers (4,5). Plasmids equipped with additional functions can be used to facilitate, for instance, coexpression of proteins assisting correct folding (6) or of tRNAs supporting transcription of rare codons (7).…”

mentioning

confidence: 99%

Comparative Transcription Profiling and In-Depth Characterization of Plasmid-Based and Plasmid-Free Escherichia coli Expression Systems under Production Conditions

Mairhofer

Scharl

Marisch

et al. 2013

Appl Environ Microbiol

View full text Add to dashboard Cite

Plasmid-based Escherichia coli BL21(DE3) expression systems are extensively used for the production of recombinant proteins. However, the combination of a high gene dosage with strong promoters exerts extremely stressful conditions on producing cells, resulting in a multitude of protective reactions and malfunctions in the host cell with a strong impact on yield and quality of the product. Here, we provide in-depth characterization of plasmid-based perturbations in recombinant protein production. A plasmid-free T7 system with a single copy of the gene of interest (GOI) integrated into the genome was used as a reference. Transcriptomics in combination with a variety of process analytics were used to characterize and compare a plasmid-free T7-based expression system to a conventional pET-plasmid-based expression system, with both expressing human superoxide dismutase in fed-batch cultivations. The plasmid-free system showed a moderate stress response on the transcriptional level, with only minor effects on cell growth. In contrast to this finding, comprehensive changes on the transcriptome level were observed in the plasmid-based expression system and cell growth was heavily impaired by recombinant gene expression. Additionally, we found that the T7 terminator is not a sufficient termination signal. Overall, this work reveals that the major metabolic burden in plasmid-based systems is caused at the level of transcription as a result of overtranscription of the multicopy product gene and transcriptional read-through of T7 RNA polymerase. We therefore conclude that the presence of high levels of extrinsic mRNAs, competing for the limited number of ribosomes, leads to the significantly reduced translation of intrinsic mRNAs. P lasmid-based expression systems have been used for the production of recombinant proteins for more than 4 decades (1, 2). They can be manipulated quickly and easily, and a variety of replicons for use in Escherichia coli have become available (3), allowing, e.g., different expression levels by using plasmids with different copy numbers (4, 5). Plasmids equipped with additional functions can be used to facilitate, for instance, coexpression of proteins assisting correct folding (6) or of tRNAs supporting transcription of rare codons (7). The dissemination of E. coli systems was further strongly supported by the availability of well-established, easy-to-use protocols from molecular manipulation to cell cultivation up to a large scale (8) and by the FDA-proven status of E. coli as a host for production of proteins for clinical use (9).However, E. coli-based production processes are still far from optimal for exploitation of the cellular system, as the expression of heterologous proteins is performed with excessive strength, leading to a rapid exhaustion of the host cell (10) and, hence, loss of yield. One prominent example is the T7 system, combining highcopy-number plasmids with an orthogonal transcription system in the form of the T7 phage RNA polymerase, an enzyme showing an average elongation ...

show abstract

Rapid optimization of gene dosage in E. coli using DIAL strains

Cited by 57 publications

References 33 publications

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

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

Principles of genetic circuit design

Comparative Transcription Profiling and In-Depth Characterization of Plasmid-Based and Plasmid-Free Escherichia coli Expression Systems under Production Conditions

Contact Info

Product

Resources

About