ABSTRACT
In recent years, the fast-growing bacterium Vibrio natriegens has gained increasing attention as it has the potential to become a next-generation chassis for synthetic biology. A wide range of genetic parts and genome engineering methods have already been developed. However, there is still a need for a well-characterized tool to effectively and gradually reduce the expression levels of native genes. To bridge this gap, we created graded-CRISPRi, a system utilizing gRNA variants that lead to varying levels of repression strength. By incorporating multiple gRNA sequences into our design, we successfully extended this concept to simultaneously repress four distinct reporter genes. Furthermore, we demonstrated the capability of using graded-CRISPRi to target native genes, thereby examining the effect of various knockdown levels on growth.
Subject(s)
RNA, Guide, CRISPR-Cas Systems , Vibrio , Vibrio/genetics , RNA, Guide, CRISPR-Cas Systems/genetics , CRISPR-Cas Systems/genetics , Gene Knockdown Techniques/methods , Synthetic Biology/methods , Gene Library , Genes, Reporter/geneticsABSTRACT
Plastic-degrading enzymes hold immense potential for eco-friendly recycling methods. However, the catalytic rates of current enzymes do not stack up against the mammoth task of degrading millions of tons of plastic waste per year. In the quest for more efficient polyethylene terephthalate (PET)-degrading enzymes, Zhang et al. report the discovery and characterization of PET40, a versatile PET-hydrolyzing esterase that is divergent from most characterized PETases. While PET40 has comparably low hydrolytic activity on PET, Zhang et al. demonstrate its broad activity on an expanded substrate pool. This sheds light on the potential ecological role of these esterases and suggests that PET might be only a recent addition to their substrate spectrum.
Subject(s)
Hydrolases , Polyethylene Terephthalates , Polyethylene Terephthalates/chemistry , Hydrolases/chemistry , Esterases , HydrolysisABSTRACT
The fast-growing bacterium Vibrio natriegens has recently gained increasing attention as a novel chassis organism for fundamental research and biotechnology. To fully harness the potential of this bacterium, highly efficient genome editing methods are indispensable to create strains tailored for specific applications. V. natriegens is able to take up free DNA and incorporate it into its genome by homologous recombination. This highly efficient natural transformation is able to mediate uptake of multiple DNA fragments, thereby allowing for multiple simultaneous edits. Here, we describe NT-CRISPR, a combination of natural transformation with CRISPR-Cas9 counterselection. In two temporally distinct steps, we first performed a genome edit by natural transformation and second, induced CRISPR-Cas9 targeting the wild type sequence, and thus leading to death of non-edited cells. Through cell killing with efficiencies of up to 99.999%, integration of antibiotic resistance markers became dispensable, enabling scarless and markerless edits with single-base precision. We used NT-CRISPR for deletions, integrations and single-base modifications with editing efficiencies of up to 100%. Further, we confirmed its applicability for simultaneous deletion of multiple chromosomal regions. Lastly, we showed that the near PAM-less Cas9 variant SpG Cas9 is compatible with NT-CRISPR and thereby broadens the target spectrum.
Subject(s)
Gene Editing , Vibrio , CRISPR-Cas Systems , DNA/genetics , Gene Editing/methods , Vibrio/geneticsABSTRACT
The capability to correctly recognize collective emotion expressions [i.e., emotional aperture (EA)] is crucial for effective social and work-related interactions. Yet, little remains known about the antecedents of this ability. The present study therefore aims to shed new light onto key aspects that may promote or diminish an individual's EA. We examine the role of age for this ability in an online sample of 181 participants (with an age range of 18-72 years, located in Germany), and we investigate agreeableness as a key contingency factor. Among individuals with lower agreeableness, on the one hand, our results indicate a curvilinear relationship between age and EA, such that EA remains at a relatively high level until these individuals' middle adulthood (with a slight increase until their late 30s) and declines afterward. Individuals with higher agreeableness, on the other hand, exhibit relatively high EA irrespective of their age. Together, these findings offer new insights for the emerging literature on EA, illustrating that specific demographic and personality characteristics may jointly shape such collective emotion recognition.
