Abstract Synthetic biology and the emerging field of biomanufacturing provide new research concepts that should be incorporated into the training of the next generation of scientists. Key elements of synthetic biology are the standardisation, modularisation, and characterisation of biological parts. Defined parts can be assembled into functional transcription units to pathways of multiple units, chromosomes, and/or whole genomes. Here, we highlight the importance of global exchange and state-of-the-art training in the Global South to empower research and reduce the gap between countries. Based on initial seed funding, we established a multidisciplinary bilateral exchange program between Germany and Colombia, including lecture series, experimental workshops, and bilateral exchanges in training and research. The elements are adaptable and our hands-on courses can be carried out with limited resources while covering aspects of synthetic biology. Importantly, this initiative will allow early-career researchers to build an international network, opening up opportunities in research collaboration and paving the way for greater equity.
Abstract Antimicrobial resistance (AMR) is a growing global threat to human health, and rapid methods for characterizing emerging antimicrobial resistance genes (ARGs) are needed. Here, we develop a semi-automated workflow using cell-free gene expression systems to measure the activity of two ARGs encoded on plasmid DNA that produce rifampicin-inactivating and gentamicin-inactivating enzymes. We validated the use of a small benchtop Myra liquid handling system compared to manual pipetting, with no statistical differences observed. After optimizing the pre-incubation time of ARGs and dispensing protocol, expression of aac(3)-IIa increased the half-maximal inhibition concentration (IC50) of gentamicin by over 150-fold, whilst arr-3 increased the IC50 of rifampicin by ~20-fold compared to controls. This methodology for rapid, semi-automated ARG characterization offers a strategy to combat AMR by assessing novel ARGs identified through genomic surveillance or profiling activity of new or derivative antibiotics.
Course-based Undergraduate Research Experiences (CUREs) have emerged as a transformative approach to science education, expanding access to authentic research opportunities beyond the traditional undergraduate research assistant (URA) training. By embedding research into a curriculum, CUREs engage a broad and diverse population of students in a classroom environment that emphasizes experimental design, data analysis, and scientific communication. However, this has been difficult to develop for fields such as plant synthetic biology due to the long timescales of plant transformation. One avenue around this problem is to utilize a recent innovation that enables high throughput and rapid screening of guide RNA (gRNA) efficacy by leveraging viral-based delivery of gRNAs. In this work, we develop and validate a CURE with undergraduate students at Colorado State University (CSU). Students worked in teams to design and test efficacy of gRNAs targeting a Cas9-based transcriptional repressor to different regions of the promoters of the three GIBBERELLIN INSENSITIVE 1 genes (GID1a, GID1b, and GID1c) in Arabidopsis thaliana. Over the semester, students generated and analysed gene expression data to understand the efficiency of twelve new gRNAs. We further validated CURE student-identified gRNAs with an URA that assessed target gene expression and phenotypic outcomes in stable transgenic lines expressing synthetic transcription factor (SynTF) constructs with the strongest gRNAs from the class. We further describe the curriculum structure to facilitate adoption at other institutions and present student-generated datasets demonstrating the utility of VipariNama-based screening for identifying effective SynTF gRNAs for plant functional genomics and engineering.
Synthetic biology and advanced genetic engineering applications rely heavily on the efficient construction of large and complex DNA sequences. Current DNA synthesis technologies have limited capacity to efficiently generate ultralong oligonucleotides for complex gene construction, particularly those with extensive repetitive motifs and uneven base distribution. Here, we report a novel platform named UCOS (Ultralong Complex Oligonucleotides Synthesis) that enables the efficient synthesis of long, complex, and challenging DNA fragments. This platform employs nonporous silica microspheres as the solid support instead of the traditional controlled pore glass solid support, full-length enrichment based on 5' flank sequence hybridization and an error-removing enzyme for correct sequence selection, substantially enhancing the fidelity of intricate, ultralong oligonucleotides. Using this approach, we successfully synthesized challenging sequences ≤600 nt in length, encompassing tandem repeats and uneven base distributions. Overall, this novel platform demonstrates exceptional efficiency and reliability in handling ultralong DNA fragments with highly repetitive and complex features. This novel platform provides a strong foundation for advancing synthetic biology and metabolic engineering, showing great potential as a powerful tool for constructing challenging genes and enabling the customized synthesis of functional genetic elements for complex genetic programmes and synthetic genomics.
The protocol for Extraction and Analysis of Small Yeast-Chromosomes (EASY-C) is a transformative, rapid, cost-effective, and user-friendly method designed for the efficient isolation of artificial synthetic mini-chromosomes (~42-52 kb) and large plasmids (~12 kb) from Saccharomyces cerevisiae wild-type and synthetic yeast strains (Sc2.0), and a range of nonconventional yeast (NCY) species. In this two-step workflow, the DNA from yeast is first extracted and transferred into bacteria, and then the circular DNA is recovered from the bacteria and subjected to downstream analysis, including long-read sequencing. The EASY-C protocol operates at small volumes (~1 mL) and requires less than 2.5 hours, allowing the use of standard commercial plasmid purification kits for bacterial plasmids. Under the tested conditions, the EASY-C methodology yielded clean DNA that could be digested and linearized prior to sequencing, resulting in a higher number of high-quality reads (~2000). The EASY-C protocol worked successfully for the extraction of a variety of constructs, including low-copy centromeric vectors (CEN/ARS), high-copy plasmids (pan/ARS), and artificial mini-chromosomes harbouring (CEN/ARS). It is also applicable to a variety of yeast species, including NCY such as Starmerella sp., Maudiozyma sp., and Kazachstania sp. Thanks to its precision, robustness, and simplicity, EASY-C equips researchers with a powerful, time-saving tool and cost-effective approach to accelerate the validation of a wide array of synthetic genetic and metabolic constructs engineered in vivo across diverse yeast species.
