Biofoundry-Scale DNA Assembly Validation Using Cost-Effective High-Throughput Long-Read Sequencing.

Biofoundries are automated high-throughput facilities specializing in the design, construction, and testing of engineered/synthetic DNA constructs (plasmids), often from genetic parts. A critical step of this process is assessing the fidelity of the assembled DNA construct to the desired design. Current methods utilized for this purpose are restriction digest or PCR followed by fragment analysis and sequencing. The Edinburgh Genome Foundry (EGF) has recently established a single-molecule sequencing quality control step using the Oxford Nanopore sequencing technology, along with a companion Nextflow pipeline and a Python package, to perform in-depth analysis and generate a detailed report. Our software enables researchers working with plasmids, including biofoundry scientists, to rapidly analyze and interpret sequencing data. In conclusion, we have created a laboratory and software protocol that validates assembled, cloned, or edited plasmids, using Nanopore long-reads, which can serve as a useful resource for the genetics, synthetic biology, and sequencing communities.

Standardization of Synthetic Biology Tools and Assembly Methods for Saccharomyces cerevisiae and Emerging Yeast Species.

As redesigning organisms using engineering principles is one of the purposes of synthetic biology (SynBio), the standardization of experimental methods and DNA parts is becoming increasingly a necessity. The synthetic biology community focusing on the engineering of Saccharomyces cerevisiae has been in the foreground in this area, conceiving several well-characterized SynBio toolkits widely adopted by the community. In this review, the molecular methods and toolkits developed for S. cerevisiae are discussed in terms of their contributions to the required standardization efforts. In addition, the toolkits designed for emerging nonconventional yeast species including Yarrowia lipolytica, Komagataella phaffii, and Kluyveromyces marxianus are also reviewed. Without a doubt, the characterized DNA parts combined with the standardized assembly strategies highlighted in these toolkits have greatly contributed to the rapid development of many metabolic engineering and diagnostics applications among others. Despite the growing capacity in deploying synthetic biology for common yeast genome engineering works, the yeast community has a long journey to go to exploit it in more sophisticated and delicate applications like bioautomation.

CRISPR-Cas9-Mediated Mutagenesis of the Rubisco Small Subunit Family in Nicotiana tabacum.

Engineering the small subunit of the key CO2-fixing enzyme Rubisco (SSU, encoded by rbcS) in plants currently poses a significant challenge, as many plants have polyploid genomes and SSUs are encoded by large multigene families. Here, we used CRISPR-Cas9-mediated genome editing approach to simultaneously knock-out multiple rbcS homologs in the model tetraploid crop tobacco (Nicotiana tabacum cv. Petit Havana). The three rbcS homologs rbcS_S1a, rbcS_S1b and rbcS_T1 account for at least 80% of total rbcS expression in tobacco. In this study, two multiplexing guide RNAs (gRNAs) were designed to target homologous regions in these three genes. We generated tobacco mutant lines with indel mutations in all three genes, including one line with a 670 bp deletion in rbcS-T1. The Rubisco content of three selected mutant lines in the T1 generation was reduced by ca. 93% and mutant plants accumulated only 10% of the total biomass of wild-type plants. As a second goal, we developed a proof-of-principle approach to simultaneously introduce a non-native rbcS gene while generating the triple SSU knockout by co-transformation into a wild-type tobacco background. Our results show that CRISPR-Cas9 is a viable tool for the targeted mutagenesis of rbcS families in polyploid species and will contribute to efforts aimed at improving photosynthetic efficiency through expression of superior non-native Rubisco enzymes in plants.

CRISPR/Cas in Arabidopsis: overcoming challenges to accelerate improvements in crop photosynthetic efficiencies.

The rapid and widespread adoption of clustered regularly interspaced short palindromic repeats (CRISPR)/Cas technologies has allowed genetic editing in plants to enter a revolutionary new era. In this mini review, we highlight the current CRISPR/Cas tools available in plants and the use of Arabidopsis thaliana as a model to guide future improvements in crop yields, such as enhancing photosynthetic potential. We also outline the current socio-political landscape for CRISPR/Cas research and highlight the growing need for governments to better facilitate research into plant genetic-editing technologies.

Female sexual advertisement reflects resource availability in twentieth-century UK society.

Evolutionary theory suggests that men and women differ in the characteristics valued in potential mates. In humans, males show a preference for physical attractiveness, whereas females seek cues that relate to resources and future earning potential. If women pursue marriage as an economic strategy, female sexual advertisement should increase during periods of poor economic conditions when the number of high-quality male partners becomes a limited resource. To test this prediction, measures of skin display and clothing tightness were taken for clothes portrayed in UK Vogue magazine from 1916 to 1999. These estimates of sexual advertisement were analyzed in relation to an index of economic prosperity (GDP), while controlling for general increases in economic conditions and sexual display over the course of the past century. The results indicate that female sexual display increases as economic conditions decline, with the level of breast display and the tightness of clothing at the waist and hips the key factors underlying this increase. Breast size and symmetry and female body form are secondary sexual characteristics that play an important role in sexual attractiveness. Since advertisement of these features increases as levels of competition for high-quality partners increases, females appear to use marriage as an economic strategy. Patterns of female fashion appear to be underpinned by evolutionary considerations relating resource availability to female reproductive success.