A Simple Enhancement for Gibson Isothermal Assembly

Rabe, Brian; Cepko, Constance L.

doi:10.1101/2020.06.14.150979

Cited by 16 publications

(11 citation statements)

References 8 publications

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“…The double stranded DNA fragment was transformed into the mc 2 155 L5::pMC1s- plrA / pNitRecET, and the resulting colonies were checked by PCR on either side of the knockout locus to confirm knockout. Vectors were assembled using Gibson cloning [ 49 ], some with the SSB enhancement [ 50 ]. After constructions and sequence confirmation, vectors were transformed into electro-competent M .…”

Section: Methodsmentioning

confidence: 99%

PlrA (MSMEG_5223) is an essential polar growth regulator in Mycobacterium smegmatis

et al. 2023

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Mycobacteria expand their cell walls at the cell poles in a manner that is not well described at the molecular level. In this study, we identify a new polar factor, PlrA, that is involved in restricting peptidoglycan metabolism to the cell poles in Mycobacterium smegmatis. We establish that only the N-terminal membrane domain of PlrA is essential. We show that depletion of plrA pheno-copies depletion of polar growth factor Wag31, and that PlrA is involved in regulating the Wag31 polar foci.

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Section: Methodsmentioning

confidence: 99%

PlrA (MSMEG_5223) is an essential polar growth regulator in Mycobacterium smegmatis

et al. 2023

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“…All DNA constructs were designed for homologous recombination at the endogenous locus with the endogenous promoter unless otherwise stated. Novel plasmids were cloned using Gibson isothermal assembly 37,38 . Briefly, fragments were amplified using PCR (NEB Q5 highfidelity DNA polymerase, M0491) from other plasmids or A. nidulans genomic DNA.…”

Section: Methodsmentioning

confidence: 99%

Woronin bodies move dynamically and bidirectionally by hitchhiking on early endosomes inAspergillus nidulans

Songster¹,

Bhuyan

Christensen

et al. 2023

Preprint

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The proper functioning of organelles depends on their intracellular localization, mediated by motor protein-dependent transport on cytoskeletal tracks. Rather than directly associating with a motor protein, peroxisomes move by hitchhiking on motile early endosomes in the filamentous fungusAspergillus nidulans. However, the cellular function of peroxisome hitchhiking is unclear. Peroxisome hitchhiking requires the protein PxdA, which is conserved within the fungal subphylum Pezizomycotina, but absent from other fungal clades. Woronin bodies are specialized peroxisomes that are also unique to the Pezizomycotina. In these fungi, multinucleate hyphal segments are separated by incomplete cell walls called septa that possess a central pore enabling cytoplasmic exchange. Upon damage to a hyphal segment, Woronin bodies plug septal pores to prevent widespread leakage. Here, we tested if peroxisome hitchhiking is important for Woronin body motility, distribution, and function inA. nidulans. We show that Woronin body proteins are present within all motile peroxisomes and hitchhike on PxdA-labeled early endosomes during bidirectional, long-distance movements. Loss of peroxisome hitchhiking by knocking out pxdA significantly affected Woronin body distribution and motility in the cytoplasm, but Woronin body hitchhiking is ultimately dispensable for septal localization and plugging.

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“…Plasmids were generated by Gibson Assembly (Gibson et al, 2009) using a homemade Gibson reaction mix based on recipes from OpenWetWare (OpenWetWare, https://openwetware.org/wiki/Gibson_Assembly) (5x Isothermal Reaction Mix [600 μL]: 300 μL 1 M Tris-HCl [pH 7.5], 30 μL 1 M MgCl2, 6 μL 100 mM dGTP, 6 μL 100 mM dATP, 6 μL 100 mM dTTP, 6 μL 100 mM dCTP, 30 μL 1 M DTT, 0.15 g PEG 8000, 60 μL 50 mM NAD + , 156 μL H 2 O) and modified by using the “enhanced” version (Rabe and Cepko, 2020) (1.33x Gibson-Assembly Master-Mix [for 25 reactions]: 100 μL 5x Isothermal Reaction-Mix, 215.32 μL H2O, 0.2 μL T5 Exonuclease [10 U/μL NEB #M0363], 6.25 μL Phusion DNA-Polymerase [2 U/μL ThermoFisher #F530L], 3.125 μL ET SSB [500 μg/ml NEB #M2401S], 50 μL Taq DNA-Ligase [40 U/μL stock NEB #M0208L]). Primers, plasmids, and strains used in this study are listed in Table S3.…”

Section: Methodsmentioning

confidence: 99%

H4K20me3 controls Ash1-mediated H3K36me3 and transcriptional silencing in facultative heterochromatin

Möller

Ridenour

Wright

et al. 2022

Preprint

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Facultative heterochromatin controls development and differentiation in many eukaryotes. In metazoans, plants, and many filamentous fungi, facultative heterochromatin is characterized by transcriptional repression and enrichment with nucleosomes that are trimethylated at histone H3 lysine 27 (H3K27me3). While loss of H3K27me3 results in derepression of transcriptional gene silencing in many species, additional up- and downstream layers of regulation are necessary to mediate control of transcription in chromosome regions enriched with H3K27me3. Here, we investigated the effects of one histone mark on histone H4, namely H4K20me3, in the fungus Zymoseptoria tritici, a globally important pathogen of wheat. Deletion of kmt5, the gene encoding the sole methyltransferase responsible for H4K20 methylation, resulted in global derepression of transcription, especially in regions of facultative heterochromatin. This finding explains our previous results where loss of H3K27me3 in kmt6 deletion strains alone was insufficient to alleviate transcriptional silencing in the same regions. Reversal of silencing in the absence of H4K20me3 not only affected genes but also a large number of novel, previously undetected, non-coding transcripts generated from regions of facultative heterochromatin on accessory chromosomes. Transcriptional activation in kmt5 deletion strains was accompanied by a complete loss of Ash1-mediated H3K36me3 and chromatin reorganization affecting H3K27me3 and H3K4me2 distribution in regions of facultative heterochromatin. Strains with a H4K20M mutation in the single histone H4 gene of Z. tritici recapitulated these chromatin changes, suggesting that H4K20me3 is essential for Ash1-mediated H3K36me3. The ∆kmt5 mutants we obtained are more sensitive to genotoxic stressors and both, ∆kmt5 and ∆ash1, showed a greatly increased rate of accessory chromosome losses. Taken together, our results provide insights into a novel, and unsuspected, mechanism controlling the assembly and maintenance of facultative heterochromatin.

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A Simple Enhancement for Gibson Isothermal Assembly

Cited by 16 publications

References 8 publications

PlrA (MSMEG_5223) is an essential polar growth regulator in Mycobacterium smegmatis

PlrA (MSMEG_5223) is an essential polar growth regulator in Mycobacterium smegmatis

Woronin bodies move dynamically and bidirectionally by hitchhiking on early endosomes inAspergillus nidulans

H4K20me3 controls Ash1-mediated H3K36me3 and transcriptional silencing in facultative heterochromatin

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