New biotechnology最新文献

ReViTA (Reverse in Vitro Transcription Assay) is a novel in vitro transcription-based method to study gene expression under the regulation of specific transcription factors. The ReViTA system uses a plasmid with a control sequence, the promoter region of the studied gene, the transcription factor of interest, and an RNA polymerase saturated with σ⁷⁰. The main objective of this study was to evaluate the method; thus, as a proof of concept, two different transcription factors were used, a transcriptional inducer, AlgR, and a repressor, LexA, from Pseudomonas aeruginosa. After the promoters were incubated with the transcription factors, the plasmid was transcribed into RNA and reverse transcribed to cDNA. Gene expression was measured using qRTPCR. Using the ReViTA plasmid, transcription induction of 55% was observed when AlgR protein was added and a 27% transcription reduction with the repressor LexA, compared with the samples without transcription factors. The results demonstrated the correct functioning of ReViTA as a novel method to study transcription factors and gene expression. Thus, ReViTA could be a rapid and accessible in vitro method to evaluate genes and regulators of various species.

Hybridization Chain Reaction (HCR) is a technique to generate a linear polymerization of oligonucleotide hairpins, used in multiple molecular biology methods. The HCR reaction is dependent on every hairpin being metastable in the absence of a triggering oligonucleotide and that every hairpin can continue the polymerization, which puts a strong demand on oligonucleotide quality. We show how further purification can greatly increase polymerization potential. It was found that a single extra PAGE-purification could greatly enhance hairpin polymerization both in solution and in situ. Purification using a ligation-based method further improved polymerization, yielding in situ immunoHCR stains at least 3.4-times stronger than a non-purified control. This demonstrates the importance of not only good sequence design of the oligonucleotide hairpins, but also the demand for high quality oligonucleotides to accomplish a potent and specific HCR.

Antibody-based cancer therapies have been evolving at a rapid pace in the pharmaceutical market. Bispecific antibody-drug conjugates that engage immune cells to target and kill cancer cells with precision have inspired the development of immunotherapy. Miniaturized antibody fragments such as diabodies, nanobodies, or single-chain variable fragments (scFvs) hold great promise as antibody-drug conjugates as they specifically target tumor tissue and can penetrate it. Here, we optimized the soluble periplasmic expression of the scFv OKT3 comprising the variable V_H and V_L domains of the mouse anti-human CD3 antibody muromonab-CD3 (trade name Orthoclone OKT3) in E. coli. By an expansion of the genetic code, we site-specifically incorporated the reactive non-canonical amino acid N^ε-((2-azidoethoxy)carbonyl)-L-lysine (AzK) into scFv OKT3 using an orthogonal pyrrolysyl-tRNA synthetase/tRNA_CUA pair. To confirm the AzK incorporation and to demonstrate the accessibility of the reactive azide group, we conjugated a fluorophore to scFv OKT3 AzK variants by copper-free strain-promoted alkyne-azide cycloaddition (‘click chemistry’). The scFv OKT3 wild type and the AzK variants bound T cells at nanomolar concentrations. In this study, a ‘ready-to-click’ scFv OKT3 was successfully developed for future applications, e.g. as controlled anti-T cell antibody-drug conjugate or bispecific T cell engager and for imaging immune T cell migration in cancers.

This study describes the cell-free biomanufacturing of a broad-spectrum antiviral protein, griffithsin (GRFT) such that it can be produced in microgram quantities with consistent purity and potency in less than 24 h. We demonstrate GRFT production using two independent cell-free systems, one plant and one microbial. Griffithsin purity and quality were verified using standard regulatory metrics. Efficacy was demonstrated in vitro against SARS-CoV-2 and HIV-1 and was nearly identical to that of GRFT expressed in vivo. The proposed production process is efficient and can be readily scaled up and deployed wherever a viral pathogen might emerge. The current emergence of viral variants of SARS-CoV-2 has resulted in frequent updating of existing vaccines and loss of efficacy for front-line monoclonal antibody therapies. Proteins such as GRFT with its efficacious and broad virus neutralizing capability provide a compelling pandemic mitigation strategy to promptly suppress viral emergence at the source of an outbreak.

Phage display technology is a powerful tool for selecting monoclonal antibodies against a diverse set of antigens. Within toxinology, however, it remains challenging to generate monoclonal antibodies against many animal toxins, as they are difficult to obtain from venom. Recombinant toxins have been proposed as a solution to overcome this challenge, but so far, few have been used as antigens to generate neutralizing antibodies. Here, we describe the recombinant expression of α-cobratoxin in E. coli and its successful application as an antigen in a phage display selection campaign. From this campaign, an scFv (single-chain variable fragment) was isolated with similar binding affinity to a control scFv generated against the native toxin. The selected scFv recognizes a structural epitope, enabling it to inhibit the interaction between the acetylcholine receptor and the native toxin in vitro. This approach represents the first entirely in vitro antibody selection strategy for generating neutralizing monoclonal antibodies against a snake toxin.

Polyhydroxyalkanoates (PHAs) are the new frontier of bioplastic production; however, research is needed to develop and characterise efficient mixed microbial communities (MMCs) for their application with a multi-feedstock approach. Here, the performance and composition of six MMCs developed from the same inoculum on different feedstocks were investigated through Illumina sequencing to understand community development and identify possible redundancies in terms of genera and PHA metabolism. High PHA production efficiencies (>80% mg COD_PHA mg^-1 COD_OA-consumed) were seen across all samples, but differences in the organic acids (OAs) composition led to different ratios of the monomers poly(3-hydroxybutyrate) (3HB) to poly(3-hydroxyvalerate) (3HV). Communities differed across all feedstocks, with enrichments in specific PHA-producing genera, but analysis of potential enzymatic activity identified a certain degree of functional redundancy, possibly leading to the general high efficiency seen in PHA production from all feedstocks. Leading PHAs producers across all feedstocks were identified in genera such as Thauera, Leadbetterella, Neomegalonema and Amaricoccus.

The limited ability of Convolutional Neural Networks to generalize to images from previously unseen domains is a major limitation, in particular, for safety-critical clinical tasks such as dermoscopic skin cancer classification. In order to translate CNN-based applications into the clinic, it is essential that they are able to adapt to domain shifts. Such new conditions can arise through the use of different image acquisition systems or varying lighting conditions. In dermoscopy, shifts can also occur as a change in patient age or occurrence of rare lesion localizations (e.g. palms). These are not prominently represented in most training datasets and can therefore lead to a decrease in performance. In order to verify the generalizability of classification models in real world clinical settings it is crucial to have access to data which mimics such domain shifts. To our knowledge no dermoscopic image dataset exists where such domain shifts are properly described and quantified. We therefore grouped publicly available images from ISIC archive based on their metadata (e.g. acquisition location, lesion localization, patient age) to generate meaningful domains. To verify that these domains are in fact distinct, we used multiple quantification measures to estimate the presence and intensity of domain shifts. Additionally, we analyzed the performance on these domains with and without an unsupervised domain adaptation technique. We observed that in most of our grouped domains, domain shifts in fact exist. Based on our results, we believe these datasets to be helpful for testing the generalization capabilities of dermoscopic skin cancer classifiers.

The development of bacterial cellulose (BC) industrialization has been seriously affected by its production. Mannose/mannan is an essential component in many biomass resources, but Komagataeibacter xylinus uses mannose in an ineffective way, resulting in waste. The aim of this study was to construct recombinant bacteria to use mannose-rich biomass efficiently as an alternative and inexpensive carbon source in place of the more commonly used glucose. This strategy aimed at modification of the mannose catabolic pathway via genetic engineering of K. xylinus ATCC 23770 strain through expression of mannose kinase and phosphomannose isomerase genes from the Escherichia coli K-12 strain. Recombinant and wild-type strains were cultured under conditions of glucose and mannose respectively as sole carbon sources. The fermentation process and physicochemical properties of BC were investigated in detail in the strains cultured in mannose media. The comparison showed that with mannose as the sole carbon source, the BC yield from the recombinant strain increased by 84%, and its tensile strength and elongation were increased 1.7 fold, while Young's modulus was increased 1.3 fold. The results demonstrated a successful improvement in BC yield and properties on mannose-based medium compared with the wild-type strain. Thus, the strategy of modifying the mannose catabolic pathway of K. xylinus is feasible and has significant potential in reducing the production costs for industrial production of BC from mannose-rich biomass.

Unspecific peroxygenase (UPO) presents a wide range of biotechnological applications. This study targets the use of by-products from bioethanol synthesis to produce UPO by Agrocybe aegerita. Solid-state and submerged fermentations (SSF and SmF) were evaluated, achieving the highest titers of UPO and laccase in SmF using vinasse as nutrients source. Optimized UPO production of 331 U/L was achieved in 50% (v:v) vinasse with an inoculum grown for 14 days. These conditions were scaled-up to a 4 L reactor, achieving a UPO activity of 265 U/L. Fungal proteome expression was analyzed before and after UPO activity appeared by shotgun mass spectrometry proteomics. Laccase, dye-decolorizing peroxidases (DyP), lectins and proteins involved in reactive oxygen species (ROS) production and control were detected (in addition to UPO). Interestingly, the metabolism of complex sugars and nitrogen sources had a different activity at the beginning and end of the submerged fermentation.

The preparation of buffer solutions used in the biopharmaceutical industry is typically performed manually by the addition of one or multiple buffering reagents to water. Recently, the adaptation of powder feeders for continuous solid feeding was demonstrated for continuous buffer preparation. However, the intrinsic characteristics of powders can change the stability of the process, due to the hygroscopic nature of some substances and humidity-induced caking and compaction behavior, but there is no simple and easy methodology available for predicting this behavior for buffer species. To predict which buffering reagents are suitable without special precautions and investigate their behavior, force displacement measurements were conducted with a customized rheometer over 18 h. While most of the eight investigated buffering reagents indicated uniform compaction, especially sodium acetate and dipotassium hydrogen phosphate (K₂HPO₄) showed a significant increase in yield stress after 2 h. Experiments conducted with a 3D printed miniaturized screw conveyor confirmed the increased yield stress measurements by visible compaction and failure of the feeding. By taking additional precautions and adjusting the design of the hopper, we demonstrated a highly linear profile of all buffering reagents over a duration of 12 and 24 h. We showed that force displacement measurements accurately predict the behavior of buffer components in continuous feeding devices for continuous buffer preparation and are a valuable tool to identify buffer components that need special precautions. Stable, precise feeding of all tested buffer components was demonstrated, highlighting the importance of identifying buffers that need a specialized setup with a rapid methodology.