Biotechnology Notes最新文献

Dengue fever is caused by any of the four serotypes of dengue viruses, DENV-1, DENV-2, DENV-3 and DENV-4 spread by mosquito bites and is important to distinguish between them due to lack of cross-protective neutralizing antibodies for each serotype. Secondary infections also put individuals at higher risk for severe dengue illness than those who have not been previously infected. Current preferred assays include reverse transcription-PCR (RT-PCR) and ELISA. To enable on-field diagnosis of dengue serotypes, the detection process would need to be simplified or at least semi-automated. A downstream detection module was conceptualized and fabricated to detect the amplified DNA from the provided PCR mix (product) of previously developed modular microfluidic chips involving sample loading, cell lysis, RNA extraction and RT-PCR. Further, to ensure accuracy, each serotype assay necessitates a positive control, negative control and test sample, which constitutes 3 separate channels for the diagnosis of just 1 serotype. In this study, a 6-channel bi-assay microfluidic chip was designed with pre-loaded diluent and cyanine dye, sample chamber for loading, sequential fluidic sample mixing, and integrated membranes for simultaneous (6-channel) fluidic manipulation from a single actuation source. Positive samples will turn the dye from blue to violet while the negative controls will remain blue. The integrated membranes provided color contrast and facilitated the manipulation of the samples to the same line of sight for simultaneous analysis, paving the way for automated color analysis via smartphone.

Synthetic biology is an engineering discipline that applies engineering principles to rationally design novel biological systems. It has the potential to contribute to solving major global challenges in a multitude of areas, from healthcare to sustainability. While the engineering biology landscape is robust and well-established in certain countries, the ecosystem and infrastructure for genetic engineering in other countries, including Slovakia, are underdeveloped. Consequently, such countries are missing the major economic and social benefits that the practical applications of the rational design of biological systems may provide. In this work, we briefly assess the status of the synthetic biology landscape in Slovakia in different areas, including research efforts, industrial participation, governmental policy, and the educational landscape. We describe the major challenges that the Slovak synthetic biology sector faces and propose a strategy that academics, policymakers, and industry could take to activate the proliferation of the Slovak synthetic biology ecosystem.

To improve the production of 3-HP with glucose as a substrate, the malonyl-CoA and propionyl-CoA pathways were coupled to regulate NADP⁺/NADPH regeneration in the recombinant E. coli. The strain Ec-AM that overexpressed the key enzymes of the malonyl-CoA pathway, acetyl CoA carboxylase (ACC) from Ustilago maydis and malonyl CoA reductase (MCR) from Chloroflexus aurantiacus, produced 0.26 g/L of 3-HP in 25-h shake flask cultivation. The strain Ec-P overexpressing the key enzyme of the propionyl-CoA pathway, propionyl-CoA dehydrogenase (PACD) from Candida rugosa, produced 0.11 g/L of 3-HP. However, 3-HP titer of the strain Ec-PAM overexpressing PACD along with ACC and MCR, via two pathways cooperation, was 1.29 g/L. The addition of biotin and bicarbonate improved the 3-HP production of the strain Ec-PAM. 3-HP titer of strain Ec-ΔY-ΔP-PAM with double deletion of ygfH (encoding propionyl-CoA: succinate-CoA transferase) and prpC (encoding methylcitrate synthase) genes reached 1.94 g/L, which was 1.5-fold higher than that of the strain Ec-PAM cultured under the same conditions.

Lipopeptides, novel biosurfactants showing versatile promising applications in enhanced oil recovery, textile industry, agriculture and daily chemical products, etc., are profoundly highlighted recently. Surfactin is one of the most typical representatives of lipopetide family. The critical micelle concentration (CMC) of surfactin is as low as 10–20 mg/L. When its concentration reaches above the CMC, different micelle structure will be formed and the surface-active performances might be changed with varied micelle morphologies. Thus, observation of the changes of surfactin micellar form at different concentrations is of great significance for its new applications. But so far, the micelle structure of surfactin (and also other lipopeptide molecules) is not reported yet, and the method for effectively observing the micelle morphology is limited as well. Here, we developed a method based on transmission electron microscopy combined with negative staining to observe the morphology of surfactin micelles, with which we can clearly observe the changes of micelle morphology of surfactin (or other lipopeptides) at different concentrations. Spherical micelles only form when the concentration of surfactin is low. With the increase in concentration, rod-shaped micelles of surfactin can form. Furthermore, complex rod-shaped-micelle-layer and big ring structure will form when the concentration of surfactin is very high.

The UK is home to a vibrant and diverse synthetic biology community. Many of its successes in research innovation and technological commercialisation can be attributed to a strong base of dedicated academics, investors, industrial leadership, and policymakers. Here, we give an overview of the organisations making up the network that have been key to these successes and the roles that they play within the different levels of the community. We start with a brief history of synthetic biology in the UK and continue by describing the progression of the societies and institutions that were set up, with particular focus on the UK's active student and entrepreneurship scene, as well as centres of research. We then contextualise the UK's growing bioeconomy, detailing government trajectories of planned innovation and how these coincide with research translation. The path to commercialisation for researchers is put into comparison to that of the US, the world leader in synthetic biology and its translation, highlighting aspects that differentiate the UK globally. Finally, we conclude with a bright outlook on the current velocity of progress and the state of the community.

As one of the most important synthetic biology elements in transcriptional regulation, promoters play irreplaceable roles in metabolic engineering. For the industrial microorganism Corynebacterium glutamicum, both the construction of a promoter library with gradient strength and the creation of ultra-strong promoters are essential for the production of target enzymes and compounds. In this work, the spacer sequence (both length and base) between the −35 and −10 regions, and the 5′-terminal untranslated region (5′UTR) were particularly highlighted to investigate their contributions to promoter strength. We constructed a series of artificially induced promoters based on the classical tac promoter using C. glutamicum ATCC13032 as the host. Here, we explored the effect of sequence length between the −35 and −10 regions on the strength of the tac promoter, and found that the mutant with 15 nt spacer length (PtacL15) was transcriptionally stronger than the classic Ptac (16 nt); subsequently, based on PtacL15, we explored the effect of the nucleotide sequence in the spacer region on transcriptional strength, and screened the strongest PtacL15m-110 (GAACAGGCTTTATCT), and PtacL15m-87 (AGTCGCTAAGACTCA); finally, we investigated the effect of the length of the 5′-terminal untranslated region (5′UTR) and screened out the optimal PtacM4 mutant with a 5′UTR length of 32 nt. Based on our new findings on the optimal spacer length (15 nt), nucleotide sequence (AGTCGCTAAGACTCA), and 5′UTR (truncated 32 nt), an ultra-strong PtacM, whose transcriptional strength was about 3.25 times that of the original Ptac, was obtained. We anticipate that these promoters with gradient transcriptional strength and the ultra-strong PtacM will play an important role in the construction of recombinant strains and industrial production.

During the current COVID-19 pandemic, the world is facing a new, highly contagious virus that suppresses innate immunity as one of its early virulence mechanisms. Therefore, finding new methods to enhance innate immunity is a promising strategy to attenuate the effects of this major global health problem. With the aim of characterizing bioactive ingredients as immune-enhancing agents, this study focuses on Abelmoschus esculentus (okra), which has several previously demonstrated bioactivities. Firstly, we investigated the immune-stimulatory effects of okra leaf ethanol extract (OLE) and okra leaf water extract (OLW) on nitric oxide (NO) production in macrophages. OLE significantly decreased nitrite accumulation in LPS-stimulated RAW 264.7 cells, indicating that it potentially inhibited NO production in a concentration-dependent manner. In contrast, OLW significantly enhanced the production of prostaglandin E₂ (PGE₂), tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), and NO in a dose-dependent manner. OLW also increased the expression levels of NO synthase (iNOS) and cyclooxygenase (COX)-2, potentially explaining the OLW-induced increase in NO and PGE₂ production. In addition, OLW stimulated the phosphorylation of mitogen-activated protein kinases (MAPKs; ERK, p38, and JNK) as well as the activation and subsequent nuclear translocation of nuclear factor κB (NF-κB). This indicated that OLW activates macrophages to secrete PGE₂, TNF-α, IL-1β, and NO, inducing iNOS and COX-2 expression via activation of the NF-κB and MAPK signaling pathways. In conclusion, our results demonstrate that OLW can effectively promote the activation of macrophages, suggesting that OLW may possess potent immunomodulatory effects and should be explored as a potential health-promoting materials to boost the immune system.

Synthetic biology has captivated scientists' imagination. It promises answers to some of the grand challenges society is facing: worsening climate crisis, insufficient food supplies for ever growing populations, and many persisting infectious and genetic diseases. While many challenges remain unaddressed, after almost two decades since its inception a number of products created by engineered biology are starting to reach the public. European scientists and entrepreneurs have been participating in delivering on the promises of synthetic biology. Associations like the European Synthetic Biology Society (EUSynBioS) play a key role in disseminating advances in the field, connecting like-minded people and promoting scientific development. In this perspective article, we review the current landscape of the synthetic biology community in Europe, discussing the state of related academic research and industry. We also discuss how EUSynBioS has helped to build bridges between professionals across the continent.

Resveratrol is a plant-derived aromatic compound with beneficial properties and it is required to develop a resveratrol production process from inexpensive biomass feedstocks. Here, we investigated the potential of Scheffersomyces stipitis, a non-conventional yeast with the capacity to utilize a wide range of sugars, to produce resveratrol from molasses, which is a by-product of sugar refineries. The S. stipitis strain metabolically engineered for resveratrol production produced resveratrol from 60 g/L mixed sugar (sucrose, glucose, and fructose), while its resveratrol titer decreased as the proportions of glucose and fructose increased. Sucrose consumption of the S. stipitis strain was clearly suppressed by the coexistence of glucose, fructose, and even ethanol. Quantitative analysis of intracellular metabolites involved in resveratrol biosynthesis using capillary electrophoresis time-of-flight mass spectrometry revealed that the composition of these sugars has a significant effect on the intracellular accumulation of glycolytic metabolites and AMP, which is an important factor involved in some cellular metabolic responses. Furthermore, the S. stipitis strain produced 1076 ± 167 mg/L of resveratrol in the fermentation with commercial sugarcane molasses (120 g/L of total sugars) as the substrate. To our knowledge, this is the first report on carbon catabolite repression in S. stipitis caused by the coexistence of sucrose, glucose, and fructose and resveratrol production from molasses. These results indicate great potential of the cost-effective resveratrol production process from molasses substrates using recombinant S. stipitis.

Fluorescent proteins are widely used molecular reporters in studying gene expression and subcellular protein localization. To enable the monitoring of transient cellular events in the model yeast Saccharomyces cerevisiae, destabilized green and cyan fluorescent proteins have been constructed. However, their co-utilization is limited by an overlap in their excitation and emission spectra. Although red fluorescent protein is compatible with both green and cyan fluorescent proteins with respect to spectra resolution, a destabilized red fluorescent protein is yet to be constructed for applications in S. cerevisiae. To realize this, we adopted a degron-fusion strategy to prompt destabilization of red fluorescent protein. Specifically, we fused two degrons derived from Cln2, a G₁-specific cyclin that mediates cell cycle transition, to the N- or C-terminus of mCherry to generate four destabilized fluorescent proteins that are soluble and functional in S. cerevisiae. Importantly, the four mCherry fluorescent proteins are highly differential with regards to fluorescence half-life and intensity, which provides a greater choice of tools available for the study of dynamic gene expression and transient cellular processes in the model yeast.