scholarly journals RepB C-terminus mutation of an ori pRi vector affects plasmid copy number inAgrobacteriumand transgene copy number in plants

2018 ◽  
Author(s):  
Zarir Vaghchhipawala ◽  
Sharon Radke ◽  
Ervin Nagy ◽  
Mary L. Russell ◽  
Susan Johnson ◽  
...  
Keyword(s):  
Transgenic Plants ◽  
Copy Number ◽  
Binary Vector ◽  
Single Copy ◽  
Plasmid Copy Number ◽  
Plant Production ◽  
Silent Mutation ◽  
Wild Type ◽  
Plasmid Copy ◽  
C Terminus

AbstractA nativerepABCreplication origin, ori pRi, was previously reported as a single copy plasmid inAgrobacterium tumefaciensand can improve the production of transgenic plants with a single copy insertion of transgenes when it is used in binary vectors forAgrobacterium-mediatedtransformation. A high copy ori pRi variant plasmid, pTF::Ri, which does not improve the frequency of single copy transgenic plants, has been reported in the literature. Sequencing the high copy pTF::RirepABCoperon revealed the presence of two mutations: one silent mutation and one missense mutation that changes a tyrosine to a histidine (Y299H) in a highly conserved area of the C-terminus of the RepB protein (RepBY299H). Reproducing these mutations in the wild-type oriRi binary vector showed thatAgrobacteriumcells with the RepBY299Hmutation grow faster on both solidified and in liquid medium, and have higher plasmid copy number as determined by ddPCR. In order to investigate the impact of the RepBY299Hmutation on transformation and quality plant production, the RepBY299Hmutated ori pRi binary vector was compared with the original wild-type ori pRi binary vector and a multi-copy oriV binary vector in canola transformation. Molecular analyses of the canola transgenic plants demonstrated that the multi-copy ori pRi with the RepBY299Hmutation inAgrobacteriumcells lost the advantage of generating high frequency single copy, backbone-free transgenic plants compared to using the single copy wild-type ori pRi binary vector.

scholarly journals RepB C-terminus mutation of a pRi-repABC binary vector affects plasmid copy number in Agrobacterium and transgene copy number in plants

PLoS ONE ◽  
2018 ◽  
Vol 13 (11) ◽  
pp. e0200972 ◽  
Author(s):  
Zarir Vaghchhipawala ◽  
Sharon Radke ◽  
Ervin Nagy ◽  
Mary L. Russell ◽  
Susan Johnson ◽  
...  
Keyword(s):  
Copy Number ◽  
Binary Vector ◽  
Plasmid Copy Number ◽  
Transgene Copy Number ◽  
Plasmid Copy ◽  
C Terminus

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.

scholarly journals Evolutionary Rescue and Drug Resistance on Multicopy Plasmids

Genetics ◽  
2020 ◽  
Vol 215 (3) ◽  
pp. 847-868
Author(s):  
Mario Santer ◽  
Hildegard Uecker
Keyword(s):  
Copy Number ◽  
Gene Dosage ◽  
Branching Processes ◽  
Plasmid Copy Number ◽  
Wild Type ◽  
Plasmid Copy ◽  
Gene Dosage Effects ◽  
Dosage Effects ◽  
Evolutionary Rescue ◽  
Establishment Probability

Bacteria often carry “extra DNA” in the form of plasmids in addition to their chromosome. Many plasmids have a copy number greater than one such that the genes encoded on these plasmids are present in multiple copies per cell. This has evolutionary consequences by increasing the mutational target size, by prompting the (transitory) co-occurrence of mutant and wild-type alleles within the same cell, and by allowing for gene dosage effects. We develop and analyze a mathematical model for bacterial adaptation to harsh environmental change if adaptation is driven by beneficial alleles on multicopy plasmids. Successful adaptation depends on the availability of advantageous alleles and on their establishment probability. The establishment process involves the segregation of mutant and wild-type plasmids to the two daughter cells, allowing for the emergence of mutant homozygous cells over the course of several generations. To model this process, we use the theory of multitype branching processes, where a type is defined by the genetic composition of the cell. Both factors—the availability of advantageous alleles and their establishment probability—depend on the plasmid copy number, and they often do so antagonistically. We find that in the interplay of various effects, a lower or higher copy number may maximize the probability of evolutionary rescue. The decisive factor is the dominance relationship between mutant and wild-type plasmids and potential gene dosage effects. Results from a simple model of antibiotic degradation indicate that the optimal plasmid copy number may depend on the specific environment encountered by the population.

scholarly journals Evolutionary rescue and drug resistance on multicopy plasmids

2019 ◽  
Author(s):  
Mario Santer ◽  
Hildegard Uecker
Keyword(s):  
Copy Number ◽  
Gene Dosage ◽  
Branching Processes ◽  
Plasmid Copy Number ◽  
Wild Type ◽  
Plasmid Copy ◽  
Gene Dosage Effects ◽  
Dosage Effects ◽  
Evolutionary Rescue ◽  
Establishment Probability

AbstractBacteria often carry “extra DNA” in form of plasmids in addition to their chromosome. Many plasmids have a copy number greater than one such that the genes encoded on these plasmids are present in multiple copies per cell. This has evolutionary consequences by increasing the mutational target size, by prompting the (transitory) co-occurrence of mutant and wild-type alleles within the same cell, and by allowing for gene dosage effects. We develop and analyze a mathematical model for bacterial adaptation to harsh environmental change if adaptation is driven by beneficial alleles on multicopy plasmids. Successful adaptation depends on the availability of advantageous alleles and on their establishment probability. The establishment process involves the segregation of mutant and wild-type plasmids to the two daughter cells, allowing for the emergence of mutant-homozygous cells over the course of several generations. To model this process, we use the theory of multi-type branching processes, where a type is defined by the genetic composition of the cell. Both factors – the number of adaptive alleles and their establishment probability – depend on the plasmid copy number, and they often do so antagonistically. We find that in the interplay of various effects, a lower or higher copy number may maximize the probability of evolutionary rescue. The decisive factor is the dominance relationship between mutant and wild-type plasmids and potential gene dosage effects. Results from a simple model of antibiotic degradation indicate that the optimal plasmid copy number may depend on the specific environment encountered by the population.

scholarly journals Segregational Drift and the Interplay between Plasmid Copy Number and Evolvability

2018 ◽  
Vol 36 (3) ◽  
pp. 472-486 ◽  
Author(s):  
Judith Ilhan ◽  
Anne Kupczok ◽  
Christian Woehle ◽  
Tanita Wein ◽  
Nils F Hülter ◽  
...  
Keyword(s):  
Copy Number ◽  
Plasmid Copy Number ◽  
Plasmid Copy

scholarly journals Plasmid copy number noise in monoclonal populations of bacteria

2010 ◽  
Vol 81 (1) ◽  
Author(s):  
Jérôme Wong Ng ◽  
Didier Chatenay ◽  
Jérôme Robert ◽  
Michael Guy Poirier
Keyword(s):  
Copy Number ◽  
Plasmid Copy Number ◽  
Plasmid Copy

scholarly journals Effects of Biofilm Growth on Plasmid Copy Number and Expression of Antibiotic Resistance Genes in Enterococcus faecalis

2013 ◽  
Vol 57 (4) ◽  
pp. 1850-1856 ◽  
Author(s):  
L. C. Cook ◽  
G. M. Dunny
Keyword(s):  
Antibiotic Resistance ◽  
Enterococcus Faecalis ◽  
Resistance Genes ◽  
Copy Number ◽  
Antibiotic Resistance Genes ◽  
Plasmid Copy Number ◽  
Plasmid Copy ◽  
Rapid Reduction ◽  
Biofilm Growth ◽  
Content Type

ABSTRACTBiofilm growth causes increased average plasmid copy number as well as increased copy number heterogeneity inEnterococcus faecaliscells carrying plasmid pCF10. In this study, we examined whether biofilm growth affected the copy number and expression of antibiotic resistance determinants for several plasmids with diverse replication systems. Four differentE. faecalisplasmids, unrelated to pCF10, demonstrated increased copy number in biofilm cells. In biofilm cells, we also observed increased transcription of antibiotic resistance genes present on these plasmids. The increase in plasmid copy number correlated with increased plating efficiency on high concentrations of antibiotics. Single-cell analysis of strains carrying two different plasmids suggested that the increase in plasmid copy number associated with biofilm growth was restricted to a subpopulation of biofilm cells. Regrowth of harvested biofilm cells in liquid culture resulted in a rapid reduction of plasmid copy number to that observed in the planktonic state. These results suggest a possible mechanism by which biofilm growth could reduce susceptibility to antibiotics in clinical settings.

scholarly journals Generating Transgenic Plants with Single-copy Insertions Using BIBAC-GW Binary Vector

10.3791/57295 ◽  
2018 ◽  
Author(s):  
Mariliis Tark-Dame ◽  
Blaise Weber ◽  
Mara de Sain ◽  
Damar Tri Anggoro ◽  
Rechien Bader ◽  
...  
Keyword(s):  
Transgenic Plants ◽  
Binary Vector ◽  
Single Copy
Sign in / Sign up

Export Citation Format

Share Document