Diego Alejandro Álvarez-Díaz, Emmanuel Valencia-Álvarez, Jorge Alonso Rivera Rivera, Aura Caterine Rengifo-Castillo, José Aldemar Usme-Ciro, Dioselina Peláez-Carvajal, Yenny Yolanda Lozano-Jiménez, Orlando Torres-Fernandez
The real-time reverse transcription-polymerase chain reaction (real-time RT-qPCR) has become a leading technique for the detection and quantification of arboviruses, including chikungunya, dengue, and zika viruses. In this study, an updated real-time RT-qPCR assay was designed and evaluated together with a synthetic positive-control chimeric RNA for the simultaneous detection and quantification of chikungunya, dengue, and Zika viruses. Amplification assays were performed to verify the construct integrity and optimal reaction/thermal cycling conditions...
June 8, 2021: Infection, Genetics and Evolution
Danh H Vu, Steven Wainaina, Mohammad J Taherzadeh, Dan Åkesson, Jorge A Ferreira
High production costs still hamper fast expansion of commercial production of polyhydroxyalkanoates (PHAs). This problem is greatly related to the cultivation medium which accounts for up to 50% of the whole process costs. The aim of this research work was to evaluate the potential of using volatile fatty acids (VFAs), derived from acidogenic fermentation of food waste, as inexpensive carbon sources for the production of PHAs through bacterial cultivation. Bacillus megaterium could assimilate glucose, acetic acid, butyric acid, and caproic acid as single carbon sources in synthetic medium with maximum PHAs production yields of 9-11%, on a cell dry weight basis...
December 2021: Bioengineered
Simon J Moore, Yonek B Hleba, Sarah Bischoff, David Bell, Karen M Polizzi, Paul S Freemont
BACKGROUND:  A key focus of synthetic biology is to develop microbial or cell-free based biobased routes to value-added chemicals such as fragrances. Originally, we developed the EcoFlex system, a Golden Gate toolkit, to study genes/pathways flexibly using Escherichia coli heterologous expression. In this current work, we sought to use EcoFlex to optimise a synthetic raspberry ketone biosynthetic pathway. Raspberry ketone is a high-value (~ £20,000 kg-1 ) fine chemical farmed from raspberry (Rubeus rubrum) fruit...
June 10, 2021: Microbial Cell Factories
Joshua Rees-Garbutt, Jake Rightmyer, Oliver Chalkley, Lucia Marucci, Claire Grierson
The minimal gene set for life has often been theorized, with at least ten produced for Mycoplasma genitalium ( M. genitalium ). Due to the difficulty of using M. genitalium in the lab, combined with its long replication time of 12-15 h, none of these theoretical minimal genomes have been tested, even with modern techniques. The publication of the M. genitalium whole-cell model provided the first opportunity to test them, simulating the genome edits in silico . We simulated minimal gene sets from the literature, finding that they produced in silico cells that did not divide...
June 10, 2021: ACS Synthetic Biology
Laura Gravelsins, Katherine Duncan, Gillian Einstein
BACKGROUND: Despite the widespread use of oral contraceptives (OCs), and the well-documented influence of estrogens, notably 17β-estradiol (E2), on cognition, research relating OCs to working memory is limited and mixed. Two factors may contribute to these mixed findings: 1) pharmacokinetics of oral contraceptives, which drive fluctuations in synthetic hormone levels; and 2) genetic polymorphisms related to dopamine degradation and working memory, which interact with E2. This research investigated whether the pharmacokinetics of oral contraceptives, in concert with the single nucleotide polymorphism (Val158Met; rs4680) of the catechol-o-methyltransferase gene (COMT), influence working memory performance...
2021: PloS One
Žana Kapustina, Aistė Jasponė, Varvara Dubovskaja, Gytis Mackevičius, Arvydas Lubys
Efficient ways to produce single-stranded DNA are of great interest for diverse applications in molecular biology and nanotechnology. In the present study, we selected T7 RNA polymerase mutants with reduced substrate specificity to employ an in vitro transcription reaction for the synthesis of chimeric DNA oligonucleotides, either individually or in pools. We performed in vitro evolution based on fluorescence-activated droplet sorting and identified mutations V783M, V783L, V689Q, and G555L as novel variants leading to relaxed substrate discrimination...
June 10, 2021: ACS Synthetic Biology
Suhad A A Al-Salihi, Ian D Bull, Raghad Al-Salhi, Paul J Gates, Kifah S M Salih, Andy M Bailey, Gary D Foster
Natural products with novel chemistry are urgently needed to battle the continued increase in microbial drug resistance. Mushroom-forming fungi are underutilized as a source of novel antibiotics in the literature due to their challenging culture preparation and genetic intractability. However, modern fungal molecular and synthetic biology tools have renewed interest in exploring mushroom fungi for novel therapeutic agents. The aims of this study were to investigate the secondary metabolites of nine basidiomycetes, screen their biological and chemical properties, and then investigate the genetic pathways associated with their production...
2021: Frontiers in Bioengineering and Biotechnology
Norazua Zakaria, Rabiatuladawiyah Ruzmi, Salmah Moosa, Norhayu Asib, Dzarifah Zulperi, Siti Izera Ismail, Muhammad Saiful Ahmad-Hamdani
Limnocharis flava (L.) Buchenau is a problematic weed in rice fields and water canals of Southeast Asia, and in Malaysia this invasive aquatic weed species has evolved multiple resistance to synthetic auxin herbicide and acetohydroxyacid synthase (AHAS) inhibitors. In this study, it was revealed that, a single nucleotide polymorphism (SNP) at amino acid position 376, where C was substituted to G at the third base of the same codon (GAC to GAG), resulting in Aspartate (Asp) substitution by Glutamate (Glu) was the contributing resistance mechanism in the L...
May 2021: Physiology and Molecular Biology of Plants: An International Journal of Functional Plant Biology
Emil D Jensen, Marcos Laloux, Beata J Lehka, Lasse E Pedersen, Tadas Jakočiūnas, Michael K Jensen, Jay D Keasling
Laboratory evolution is a powerful approach to search for genetic adaptations to new or improved phenotypes, yet either relies on labour-intensive human-guided iterative rounds of mutagenesis and selection, or prolonged adaptation regimes based on naturally evolving cell populations. Here we present CRISPR- and RNA-assisted in vivo directed evolution (CRAIDE) of genomic loci using evolving chimeric donor gRNAs continuously delivered from an error-prone T7 RNA polymerase, and directly introduced as RNA repair donors into genomic targets under either Cas9 or dCas9 guidance...
June 9, 2021: Nucleic Acids Research
Yuki Soma, Masatomo Takahashi, Yuri Fujiwara, Tamaki Shinohara, Yoshihiro Izumi, Taizo Hanai, Takeshi Bamba
Dynamic metabolic engineering that harnesses synthetic biological tools is a next-generation strategy for microbial chemical and fuel production. We previously reported a synthetic quorum sensing system combined with a metabolic toggle switch (QS-MTS) in E. coli . It autonomously redirected endogenous metabolic flux toward the synthetic metabolic pathway and improved biofuel production. However, its functions and effects on host metabolism were attenuated by induction timing delay. Here, we redesigned the QS-MTS to stabilize QS signaling efficiency and metabolic regulation...
June 9, 2021: ACS Synthetic Biology
Liyuan Chen, Hai Liu, Li Wang, Xiaoming Tan, Shihui Yang
Due to its robustness to environmental stresses and fast growth, Synechococcus elongatus UTEX2973 is developed as a new model for researches on cyanobacterial molecular biology and biotechnology. However, systematic genetic modifications of S. elongatus UTEX2973 were hindered by the lack of effective genetic manipulation tools, especially available counter-selection markers. Here, six synthetic counter-selection markers (SCOMs) were assembled by fusing six toxin genes from either Escherichia coli or cyanobacteria with a theophylline-inducible promoter...
June 9, 2021: Applied Microbiology and Biotechnology
Malgorzata E Futyma, Yaojie Guo, Casper Hoeck, Jakob B Hoof, Charlotte H Gotfredsen, Uffe H Mortensen, Thomas O Larsen
In recent years, there has been an increasing demand for the replacement of synthetic food colorants with naturally derived alternatives. Filamentous fungi are prolific producers of secondary metabolites including polyketide-derived pigments, many of which have not been fully characterized yet. During our ongoing investigations of black aspergilli, we noticed that Aspergillus homomorphus turned yellow when cultivated on malt extract agar plates. Chemical discovery guided by UV and MS led to the isolation of two novel yellow natural products, and their structures were elucidated as aromatic α-pyrones homopyrones A (1) and B (2) by HRMS and NMR...
June 9, 2021: Applied Microbiology and Biotechnology
Rodrigo Ibarra-Chávez, Mads Frederik Hansen, Rafael Pinilla-Redondo, Kimberley D Seed, Urvish Trivedi
The arms race between (bacterio)phages and their hosts are recognised hotspots for genome evolution. Indeed, phages and their components have historically paved the way for many molecular biology techniques and biotech applications. Further exploration into their complex lifestyles has revealed that phages are often parasitised by distinct types of hyperparasitic mobile genetic elements. These so-called phage satellites exploit phages to ensure their own propagation and horizontal transfer into new bacterial hosts, and their prevalence and peculiar lifestyle has caught the attention of many researchers...
June 9, 2021: FEMS Microbiology Reviews
Katherine M Elston, Sean P Leonard, Peng Geng, Sarah B Bialik, Elizabeth Robinson, Jeffrey E Barrick
Insects are an incredibly diverse group of animals with species that benefit and harm natural ecosystems, agriculture, and human health. Many insects have consequential associations with microbes: bacterial symbionts may be embedded in different insect tissues and cell types, inherited across insect generations, and required for insect survival and reproduction. Genetically engineering insect symbionts is key to understanding and harnessing these associations. We summarize different types of insect-bacteria relationships and review methods used to genetically modify endosymbiont and gut symbiont species...
June 5, 2021: Trends in Microbiology
Stina George Fernandes, Prachi Shah, Ekta Khattar
DNA integrity is continuously challenged by intrinsic cellular processes and environmental agents. To overcome this genomic damage, cells have developed multiple signaling pathways collectively named as DNA damage response (DDR) and composed of three components: (i) sensor proteins, which detect DNA damage, (ii) mediators that relay the signal downstream and recruit the repair machinery, and (iii) the repair proteins, which restore the damaged DNA. A flawed DDR and failure to repair the damage lead to the accumulation of genetic lesions and increased genomic instability, which is recognized as a hallmark of cancer...
June 7, 2021: Anti-cancer Agents in Medicinal Chemistry
Aset Khakimzhan, David Garenne, Benjamin Tickman, Jason Fontana, James Carothers, Vincent Noireaux
It is established that for CRISPR-Cas9 genetic guide RNAs with 17-20bp long spacer sequences are optimal for accurate target binding and cleavage. In this work, we perform cell-free CRISPRa (CRISPR activation) and CRISPRi (CRISPR inhibition) experiments to demonstrate the existence of a complex dependence of CRISPR-Cas9 binding as a function of the spacer length and complementarity. Our results show that significantly truncated or mismatched spacer sequences can form stronger guide-target bonds than the conventional 18-20bp long spacers...
June 8, 2021: Physical Biology
Yang Zhang, Xiaohui Song, Yumeng Lai, Qiwen Mo, Jifeng Yuan
Energy crisis and global climate change have driven an increased effort toward biofuel synthesis from renewable feedstocks. Herein, purple nonsulfur photosynthetic bacterium (PNSB) of Rhodobacter capsulatus was explored as a platform for high-titer production of a terpene-based advanced biofuel-bisabolene. A multilevel engineering strategy such as promoter screening, improving the NADPH availability, strengthening the precursor supply, suppressing the side pathways, and introducing a heterologous mevalonate pathway, was used to improve the bisabolene titer in R...
June 8, 2021: ACS Synthetic Biology
Dengwei Lei, Zetian Qiu, Jihua Wu, Bin Qiao, Jianjun Qiao, Guang-Rong Zhao
The monoterpene alcohols acyclic nerol and bicyclic borneol are widely applied in the food, cosmetic, and pharmaceutical industries. The emerging synthetic biology enables microbial production to be a promising alternative for supplying monoterpene alcohols in an efficient and sustainable approach. In this study, we combined metabolic and plant monoterpene synthase engineering to improve the de novo production of nerol and borneol in prene-overproducing Escherichia coli . We engineered the growth-orthogonal neryl diphosphate (NPP) as the universal precursor of monoterpene alcohol biosynthesis and coexpressed nerol synthase (GmNES) from Glycine max to generate nerol or coexpressed the truncated bornyl diphosphate synthase (LdtBPPS) from Lippia dulcis for borneol production...
June 8, 2021: ACS Synthetic Biology
Hasan Baig, Pedro Fontanarossa, Vishwesh Kulkarni, James McLaughlin, Prashant Vaidyanathan, Bryan Bartley, Shyam Bhakta, Swapnil Bhatia, Mike Bissell, Kevin Clancy, Robert Sidney Cox, Angel Goñi Moreno, Thomas Gorochowski, Raik Grunberg, Jihwan Lee, Augustin Luna, Curtis Madsen, Goksel Misirli, Tramy Nguyen, Nicolas Le Novere, Zachary Palchick, Matthew Pocock, Nicholas Roehner, Herbert Sauro, James Scott-Brown, John T Sexton, Guy-Bart Stan, Jeffrey J Tabor, Logan Terry, Marta Vazquez Vilar, Christopher A Voigt, Anil Wipat, David Zong, Zach Zundel, Jacob Beal, Chris Myers
People who are engineering biological organisms often find it useful to communicate in diagrams, both about the structure of the nucleic acid sequences that they are engineering and about the functional relationships between sequence features and other molecular species. Some typical practices and conventions have begun to emerge for such diagrams. The Synthetic Biology Open Language Visual (SBOL Visual) has been developed as a standard for organizing and systematizing such conventions in order to produce a coherent language for expressing the structure and function of genetic designs...
June 7, 2021: Journal of Integrative Bioinformatics
Josef Huntington, Manendra Pachauri, Hashim Ali, Mauro Giacca
There is an impelling need to develop new therapeutics for myocardial infarction and heart failure. A novel and exciting therapeutic possibility is to achieve cardiac regeneration through the stimulation of the endogenous capacity of cardiomyocytes to proliferate. Proof-of-concept evidence of microRNA-induced cardiac regeneration is available in both small and large animals using viral vectors. However, a clinically more applicable strategy is the development of lipid-mediated nanotechnologies for the administration of RNA therapeutics as synthetic molecules...
June 4, 2021: Current Opinion in Genetics & Development
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