17 Matching Annotations
  1. Jan 2021
    1. 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)
  2. Dec 2020
    1. 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.

    1. 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

  3. Nov 2020
    1. 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.

    2. pWV01 is a cryptic plasmid originally purified from Streptococcus cremoris [7]. Its RCR origin has been used to create over 20 cloning vectors
    1. 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
    2. 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

    3. 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

    1. 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?

    1. 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
    1. 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
    1. 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
  4. Mar 2020
    1. Mobilizable plasmids carry only the relaxosomal components oriT, a relaxase gene, and one or more nicking auxiliary proteins
  5. Jan 2020
    1. OCTOPUS, a light-weight, cost-effective, and robust method for full-plasmid sequence verification using next-generation sequencing
  6. Dec 2019
    1. 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
  7. Sep 2019
    1. 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