RESUMO
CRISPR SWAPnDROP extends the limits of genome editing to large-scale in-vivo DNA transfer between bacterial species. Its modular platform approach facilitates species specific adaptation to confer genome editing in various species. In this study, we show the implementation of the CRISPR SWAPnDROP concept for the model organism Escherichia coli, the fast growing Vibrio natriegens and the plant pathogen Dickeya dadantii. We demonstrate the excision, transfer and integration of large chromosomal regions between E. coli, V. natriegens and D. dadantii without size-limiting intermediate DNA extraction. CRISPR SWAPnDROP also provides common genome editing approaches comprising scarless, marker-free, iterative and parallel insertions and deletions. The modular character facilitates DNA library applications, and recycling of standardized parts. Its multi-color scarless co-selection system significantly improves editing efficiency and provides visual quality controls throughout the assembly and editing process.
Assuntos
Sistemas CRISPR-Cas , Edição de Genes , Sistemas CRISPR-Cas/genética , DNA/genética , Escherichia coli/genética , Terapia Genética , Genoma Bacteriano/genéticaRESUMO
Transcription, the first step to gene expression, is a central coordination process in all living matter. Besides a plethora of regulatory mechanisms, the promoter architecture sets the foundation of expression strength, timing and the potential for further regulatory modulation. In this study, we investigate the effects of promoter spacer length and sequence composition on strength and supercoiling sensitivity in bacteria. Combining transcriptomics data analysis and standardized synthetic promoter libraries, we exclude effects of specific promoter sequence contexts. Analysis of promoter activity shows a strong variance with spacer length and spacer sequence composition. A detailed study of the spacer sequence composition under selective conditions reveals an extension to the -10 region that enhances RNAP binding but damps promoter activity. Using physiological changes in DNA supercoiling levels, we link promoter supercoiling sensitivity to overall spacer GC-content. Time-resolved promoter activity screens, only possible with a novel mild treatment approach, reveal strong promoter timing potentials solely based on DNA supercoiling sensitivity in the absence of regulatory sites or alternative sigma factors.
Assuntos
DNA Bacteriano/genética , DNA Super-Helicoidal/genética , Escherichia coli/genética , Composição de Bases , DNA Bacteriano/química , DNA Super-Helicoidal/química , Escherichia coli/química , Regiões Promotoras Genéticas , TranscriptomaRESUMO
Modern cloning solutions are gradually replacing classical cloning methods. Current systems make use of libraries with predefined DNA parts that are joined by Golden-Gate reactions. However, these systems still suffer from specific inflexibilities and the lack of inter-compatibility. Here, we present Flexible Modular Cloning (MoCloFlex) which overcomes this inflexibility by introducing a set of linker- and position-vectors allowing free unit arrangement. Our system, therefore, provides a convenient way to design and build custom plasmids, and iterative assembly of large constructs. To support standardization in synthetic biology, MoCloFlex is compatible with the well-established Modular Cloning standard. Here, we present and characterize MoCloFlex for various applications with up to 12 fragments in a single restriction-ligation reaction.
RESUMO
Although the formation of rod-shaped chromosomes is vital for the correct segregation of eukaryotic genomes during cell divisions, the molecular mechanisms that control the chromosome condensation process have remained largely unknown. Here, we identify the C2H2 zinc-finger transcription factor Zas1 as a key regulator of mitotic condensation dynamics in a quantitative live-cell microscopy screen of the fission yeast Schizosaccharomyces pombe By binding to specific DNA target sequences in their promoter regions, Zas1 controls expression of the Cnd1 subunit of the condensin protein complex and several other target genes, whose combined misregulation in zas1 mutants results in defects in chromosome condensation and segregation. Genetic and biochemical analysis reveals an evolutionarily conserved transactivation domain motif in Zas1 that is pivotal to its function in gene regulation. Our results suggest that this motif, together with the Zas1 C-terminal helical domain to which it binds, creates a cis/trans switch module for transcriptional regulation of genes that control chromosome condensation.
Assuntos
Proteínas de Ciclo Celular/genética , Cromossomos Fúngicos/genética , Mitose/genética , Proteínas de Schizosaccharomyces pombe/genética , Adenosina Trifosfatases/genética , Proteínas de Ligação a DNA/genética , Regulação Fúngica da Expressão Gênica/genética , Complexos Multiproteicos/genética , Mutação , Schizosaccharomyces/genéticaRESUMO
Condensins associate with DNA and shape mitotic chromosomes. Condensins are enriched nearby highly expressed genes during mitosis, but how this binding is achieved and what features associated with transcription attract condensins remain unclear. Here, we report that condensin accumulates at or in the immediate vicinity of nucleosome-depleted regions during fission yeast mitosis. Two transcriptional coactivators, the Gcn5 histone acetyltransferase and the RSC chromatin-remodelling complex, bind to promoters adjoining condensin-binding sites and locally evict nucleosomes to facilitate condensin binding and allow efficient mitotic chromosome condensation. The function of Gcn5 is closely linked to condensin positioning, since neither the localization of topoisomerase II nor that of the cohesin loader Mis4 is altered in gcn5 mutant cells. We propose that nucleosomes act as a barrier for the initial binding of condensin and that nucleosome-depleted regions formed at highly expressed genes by transcriptional coactivators constitute access points into chromosomes where condensin binds free genomic DNA.
