Rapid pathogen sensing remains a pressing issue today since conventional identification methodsare tedious, cost intensive and time consuming, typically requiring from 48 to 72 h. In turn, chip based technologies, such as microarrays and microfluidic biochips, offer real alternatives capable of filling this technological gap. In particular microfluidic biochips make the development of fast, sensitive and portable diagnostic tools possible, thus promising rapid and accurate detection of a variety of pathogens. This paper will provide a broad overview of the novel achievements in the field of pathogen sensing by focusing on methods and devices that compliment microfluidics.
Modulating resource allocation in bacteria to redirect metabolic building blocks to the formation of recombinant proteins rather than biomass formation remains a grand challenge in biotechnology. Here, we present a novel approach for improved recombinant protein production (RPP) using Escherichia coli (E. coli) by decoupling recombinant protein synthesis from cell growth. We show that cell division and host mRNA transcription can be successfully inhibited by coexpression of a bacteriophagederived E. coli RNA polymerase (RNAP) inhibitor peptide and that genes overtranscribed by the orthogonal T7 RNAP can finally account to >55% of cell dry mass (CDM). This RNAP inhibitor peptide binds the E. coli RNAP and therefore prevents σ-factor 70 mediated formation of transcriptional qualified open promoter complexes. Thereby, the transcription of σ-factor 70 driven host genes is inhibited, and metabolic resources can be exclusively utilized for synthesis of the protein of interest (POI). Here, we mimic the late phase of bacteriophage infection by coexpressing a phage-derived xenogeneic regulator that reprograms the host cell and thereby are able to significantly improve RPP under industrial relevant fed-batch process conditions at bioreactor scale. We have evaluated production of several different recombinant proteins at different scales (from microscale to 20 L fed-batch scale) and have been able to improve total and soluble proteins yields up to 3.4fold in comparison to the reference expression system E. coli BL21(DE3). This novel approach for growth-decoupled RPP has profound implications for biotechnology and bioengineering and helps to establish more cost-effective and generic manufacturing processes for biologics and biomaterials.
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 ...
The phage-derived T7 RNA polymerase is the most prominent orthogonal transcriptions system used in the field of synthetic biology. However, gene expression driven by T7 RNA polymerase is prone to read-through transcription due to contextuality of the T7 terminator. The native T7 terminator has a termination efficiency of approximately 80% and therefore provides insufficient insulation of the expression unit. By using a combination of a synthetic T7 termination signal with two well-known transcriptional terminators (rrnBT1 and T7), we have been able to increase the termination efficiency to 99%. To characterize putative effects of an enhanced termination signal on product yield and process stability, industrial-relevant fed batch cultivations have been performed. Fermentation of a E. coli HMS174(DE3) strain carrying a pET30a derivative containing the improved termination signal showed a significant decrease of plasmid copy number (PCN) and an increase in total protein yield under standard conditions.
Background: Antibody tests are essential tools to investigate humoral immunity following SARS-CoV-2 infection or vaccination. While first-generation antibody tests have primarily provided qualitative results, accurate seroprevalence studies and tracking of antibody levels over time require highly specific, sensitive and quantitative test setups. Methods: We have developed two quantitative, easy-to-implement SARS-CoV-2 antibody tests, based on the spike receptor binding domain and the nucleocapsid protein. Comprehensive evaluation of antigens from several biotechnological platforms enabled the identification of superior antigen designs for reliable serodiagnostic. Cut-off modelling based on unprecedented large and heterogeneous multicentric validation cohorts allowed us to define optimal thresholds for the tests' broad applications in different aspects of clinical use, such as seroprevalence studies and convalescent plasma donor qualification. Findings: Both developed serotests individually performed similarly-well as fully-automated CE-marked test systems. Our described sensitivity-improved orthogonal test approach assures highest specificity (99.8%); thereby enabling robust serodiagnosis in low-prevalence settings with simple test formats. The inclusion of a calibrator permits accurate quantitative monitoring of antibody concentrations in samples collected at different time points during the acute and convalescent phase of COVID-19 and disclosed antibody level thresholds that correlate well with robust neutralization of authentic SARS-CoV-2 virus. Interpretation: We demonstrate that antigen source and purity strongly impact serotest performance. Comprehensive biotechnology-assisted selection of antigens and in-depth characterisation of the assays allowed us to overcome limitations of simple ELISA-based antibody test formats based on chromometric reporters, to yield comparable assay performance as fully-automated platforms.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
customersupport@researchsolutions.com
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
This site is protected by reCAPTCHA and the Google Privacy Policy and Terms of Service apply.
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.