- May 2023
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journals.asm.org journals.asm.org
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was digested with PstI (TaKaRa, Japan) to prevent the copy number from being underestimated (66).
Why does digestion improve the estimation of plasmid vs the chromosome? I would assume the chromosome is more tightly supercoiled and inaccessible unless digested right?
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- Mar 2021
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jcm.asm.org jcm.asm.org
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use of plasmid DNA enabled lower depth sequencing, and assemblies sufficient for full antimicrobial resistance gene annotation were obtained with as few as 2,000 to 5,000 reads, which could be acquired in 20 min of sequencing
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- Jan 2021
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www.ncbi.nlm.nih.gov www.ncbi.nlm.nih.gov
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RSF1010 is maintained at a copy-number of about 10-12 per chromosome in E coli, P. aeruginosa and S. enterica sv.Typhimurium (Frey and Bagdasarian, 1989)
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- Dec 2020
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www-sciencedirect-com.ezproxy.rice.edu www-sciencedirect-com.ezproxy.rice.edu
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In this study, we have quantitated yields of low copy and single copy number plasmid DNAs after growth of cells in four widely used broths (SB, SOC, TB, and 2xYT) and compared results to those obtained with LB, the most common E. coli cell growth medium
TB (terrific broth) consistently generated the greatest amount of plasmid DNA, in agreement with its ability to produce higher cell titers. The superiority of TB was primarily due to its high levels of yeast extract (24 g/L) and was independent of glycerol, a unique component of this broth. Interestingly, simply preparing LB with similarly high levels of yeast extract (LB24 broth) resulted in plasmid yields that were equivalent to those of TB.
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www.zymoresearch.com www.zymoresearch.com
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www.ncbi.nlm.nih.gov www.ncbi.nlm.nih.gov
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The pBHR1 plasmid is a derivative of the small (2.6-kb), mobilizable broad-host-range plasmid pBBR1, which was isolated from the gram-negative bacterium Bordetella bronchiseptica
pBBR1
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- Nov 2020
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journals.plos.org journals.plos.org
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Replication begins
Rolling circle replication overview
Replication begins when the Rep protein, which is encoded on the plasmid (ORF A), recognizes a specific site on the plasmid (double-strand origin, or DSO) and catalyzes the nicking of one DNA strand. The Rep protein remains bound to the 5′ phosphate after the nicking action. The newly released 3′ hydroxyl on the opposite end serves as a primer for DNA synthesis. The host DNA polymerase uses the unnicked circular strand as a template, so that a single replication fork moves around a plasmid until it regenerates the DSO. A second copy of Rep protein catalyzes the cleavage of the newly formed DSO, effectively releasing a single stranded copy of the plasmid. In the absence of Rep, the replication fork continues to move around the template, forming a single stranded concatemer. The single strand origin (SSO), a non-coding element that forms extensive secondary structure, is required for synthesis of the lagging strand. SSO sequences vary considerably among different RCR plasmids, but are extremely important for robust replication of the plasmid in the cell [10]. Here we describe the engineering of the pWV01 RCR origin to create pBAV1K-T5, a very broad-host range expression vector.
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pWV01 is a cryptic plasmid originally purified from Streptococcus cremoris [7]. Its RCR origin has been used to create over 20 cloning vectors
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www-nature-com.ezproxy.rice.edu www-nature-com.ezproxy.rice.edu
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Copy numbers of the oriV region and gfp gene on the plasmid relative to a chromosomal gene were determined by qPCR. The chromosomal gene dapA was set up as the reference gene
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IS186-mediated integration of the plasmid into the chromosome or deletion of these accessory genes from an evolved plasmid that remained capable of self-replication conferred greater fitness benefits than SP formation
Can we say this is because of the combined fitness benefit
- by avoiding maintenance of a plasmid and
- lower expression of proteins?
Especially considering that there is expression of the accessory genes (ex: GFP in fig.2e)-> so the protein level fitness burden still exists, albeit at a lower extent.
How much of this burden is attributed to keeping a plasmid around?, maybe this could be tested with a low copy pSC101 type plasmid or by deleting all the accessory genes and repeating the evolution experiment to specifically look for the integrants this time
- by avoiding maintenance of a plasmid and
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The deletions leading to SP formation were nearly always flanked by short, near-perfect sequence homologies with lengths of 7–15 base pairs (Supplementary Table 1), suggesting that SPs may form through RecA-independent processes
deletion in plasmid fragments evolution
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microbialcellfactories.biomedcentral.com microbialcellfactories.biomedcentral.com
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Designed probes were modified at the 5′ end with the fluorophore FAM for the reference gene cysG and HEX for the plasmid marker oriT, and furthermore modified at the 3′ end with the quencher BHQ-1
plasmid copy number qPCR assay.
Why cysG: Siroheme synthase gene?
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www.frontiersin.org www.frontiersin.org
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Among the fully sequenced BHR plasmids, most are classified as the well-known incompatibility groups, such as IncP-1 (41 plasmids), IncW (5 plasmids), and IncU (4 plasmids), based on the backbone genes
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www-sciencedirect-com.ezproxy.rice.edu www-sciencedirect-com.ezproxy.rice.edu
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broad-host-range plasmid RSF1010 is a member of the IncQ group of plasmids, which stably replicate in a wide variety of gram-negative and gram-positive bacteria, including E. coli and several strains of cyanobacteria
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jb.asm.org jb.asm.org
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Conditionally replicating plasmids of the R6K family (IncX) are very often used to deliver gene-inactivating elements in enteric bacteria (11, 28, 38). The replication of these plasmids requires the pir-encoded Π protein, which is usually provided in trans in the donor strain
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- Mar 2020
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mmbr.asm.org mmbr.asm.org
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Mobilizable plasmids carry only the relaxosomal components oriT, a relaxase gene, and one or more nicking auxiliary proteins
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- Jan 2020
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OCTOPUS, a light-weight, cost-effective, and robust method for full-plasmid sequence verification using next-generation sequencing
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- Dec 2019
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academic.oup.com academic.oup.com
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plasmid-based circuits suffer from multiple limitations: high intercellular variation in gene expression, genetic instability from random partitioning of plasmids during cell division, and plasmid loss in environments for which antibiotic use could disrupt native microbial communities or is economically infeasible
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- Sep 2019
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www.biorxiv.org www.biorxiv.org
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Plasmids (IncP/N/W)with 373short and rigid pili only transfer efficiently on solid surfaces, unlike those with longand flexible 374pili (IncF/H/T/J),capable of transferring equally well in liquidand on solid surfaces[35,36]
Plasmid group vs pili length
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