scholarly journals Plasmid stability in immobilized and free recombinant Escherichia coli JM105(pKK223-200): importance of oxygen diffusion, growth rate, and plasmid copy number.

1987 ◽  
Vol 53 (7) ◽  
pp. 1548-1555 ◽  
Author(s):  
P de Taxis du Poët ◽  
Y Arcand ◽  
R Bernier ◽  
J N Barbotin ◽  
D Thomas
Keyword(s):  
Escherichia Coli ◽  
Growth Rate ◽  
Oxygen Diffusion ◽  
Copy Number ◽  
Plasmid Stability ◽  
Plasmid Copy Number ◽  
Recombinant Escherichia Coli ◽  
Plasmid Copy ◽  
Diffusion Growth

scholarly journals Absolute and relative QPCR quantification of plasmid copy number in Escherichia coli

2006 ◽  
Vol 123 (3) ◽  
pp. 273-280 ◽  
Author(s):  
Changsoo Lee ◽  
Jaai Kim ◽  
Seung Gu Shin ◽  
Seokhwan Hwang
Keyword(s):  
Escherichia Coli ◽  
Copy Number ◽  
Plasmid Copy Number ◽  
Plasmid Copy ◽  
Qpcr Quantification

scholarly journals Cloning and functional characterization of Komagataella phaffii centromeres by a color-based plasmid stability assay

2018 ◽  
Author(s):  
Luiza Cesca Piva ◽  
Janice Lisboa De Marco ◽  
Lidia Maria Pepe de Moraes ◽  
Viviane Castelo Branco Reis ◽  
Fernando Araripe Gonçalves Torres
Keyword(s):  
Copy Number ◽  
Plasmid Stability ◽  
Functional Characterization ◽  
Plasmid Copy Number ◽  
Plasmid Copy ◽  
Komagataella Phaffii ◽  
High Transformation ◽  
Colony Color ◽  
Stable Vectors

AbstractThe yeast Komagataella phaffii is widely used as a microbial host for heterologous protein production. However, molecular tools for this yeast are basically restricted to a few integrative and replicative plasmids. Four sequences that have recently been proposed as the K. phaffii centromeres could be used to develop a new class of mitotically stable vectors. In this work we designed a color-based genetic assay to investigate genetic stability in K. phaffii. Plasmids bearing K. phaffii centromeres and the ADE3 marker were evaluated in terms of mitotic stability in an ade2/ade3 auxotrophic strain which allows plasmid screening through colony color. Plasmid copy number was verified through qPCR. Our results confirmed that the centromeric plasmids were maintained at low copy number as a result of typical chromosome-like segregation during cell division. These features, combined with high transformation efficiency and in vivo assembly possibilities, prompt these plasmids as a new addition to the K. phaffii genetic toolbox.

scholarly journals Restoration of wild-type motility to flagellin-knockout Escherichia coli

2019 ◽  
Author(s):  
Nicholas M. Thomson ◽  
Mark J. Pallen
Keyword(s):  
Escherichia Coli ◽  
Copy Number ◽  
Plasmid Copy Number ◽  
Major Constituent ◽  
Strongly Correlated ◽  
Wild Type ◽  
Plasmid Copy ◽  
Inducible Promoters ◽  
E Coli ◽  
Flagellar Filament

AbstractFlagellin is the major constituent of the flagellar filament and faithful restoration of wild-type motility to flagellin mutants may be beneficial for studies of flagellar biology and biotechnological exploitation of the flagellar system. Therefore, we explored the restoration of motility by flagellin expressed from a variety of combinations of promoter, plasmid copy number and induction strength. Motility was only partially restored using the tightly regulated rhamnose promoter, but wild-type motility was achieved with the T5 promoter, which, although leaky, allowed titration of induction strength. Motility was little affected by plasmid copy number when dependent on inducible promoters. However, plasmid copy number was important when expression was controlled by the native E. coli flagellin promoter. Motility was poorly correlated with flagellin transcription levels, but strongly correlated with the amount of flagellin associated with the flagellar filament, suggesting that excess monomers are either not exported or not assembled into filaments. This study provides a useful reference for further studies of flagellar function and a simple blueprint for similar studies with other proteins.

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