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 Genetic Alteration of Mycobacterium smegmatis To Improve Mycobacterium-Mediated Transfer of Plasmid DNA into Mammalian Cells and DNA Immunization

2007 ◽  
Vol 75 (10) ◽  
pp. 4804-4816 ◽  
Author(s):  
Yongkai Mo ◽  
Natalie M. Quanquin ◽  
William H. Vecino ◽  
Uma Devi Ranganathan ◽  
Lydia Tesfa ◽  
...  
Keyword(s):  
Gene Transfer ◽  
Mycobacterium Smegmatis ◽  
Copy Number ◽  
Mammalian Cells ◽  
Mononuclear Cells ◽  
Fold Increase ◽  
Plasmid Copy Number ◽  
Plasmid Copy ◽  
J774 Cells

ABSTRACT Mycobacteria target and persist within phagocytic monocytes and are strong adjuvants, making them attractive candidate vectors for DNA vaccines. We characterized the ability of mycobacteria to deliver transgenes to mammalian cells and the effects of various bacterial chromosomal mutations on the efficiency of transfer in vivo and in vitro. First, we observed green fluorescent protein expression via microscopy and fluorescence-activated cell sorting analysis after infection of phagocytic and nonphagocytic cell lines by Mycobacterium smegmatis or M. bovis BCG harboring a plasmid encoding the fluorescence gene under the control of a eukaryotic promoter. Next, we compared the efficiencies of gene transfer using M. smegmatis or BCG containing chromosomal insertions or deletions that cause early lysis, hyperconjugation, or an increased plasmid copy number. We observed a significant—albeit only 1.7-fold—increase in the level of plasmid transfer to eukaryotic cells infected with M. smegmatis hyperconjugation mutants. M. smegmatis strains that overexpressed replication proteins (Rep) of pAL5000, a plasmid whose replicon is incorporated in many mycobacterial constructs, generated a 10-fold increase in plasmid copy number and 3.5-fold and 3-fold increases in gene transfer efficiency to HeLa cells and J774 cells, respectively. Although BCG strains overexpressing Rep could not be recovered, BCG harboring a plasmid with a copy-up mutation in oriM resulted in a threefold increase in gene transfer to J774 cells. Moreover, M. smegmatis strains overexpressing Rep enhanced gene transfer in vivo compared with a wild-type control. Immunization of mice with mycobacteria harboring a plasmid (pgp120h E) encoding human immunodeficiency virus gp120 elicited gp120-specific CD8 T-cell responses among splenocytes and peripheral blood mononuclear cells that were up to twofold (P < 0.05) and threefold (P < 0.001) higher, respectively, in strains supporting higher copy numbers. The magnitude of these responses was approximately one-half of that observed after intramuscular immunization with pgp120h E. M. smegmatis and other nonpathogenic mycobacteria are promising candidate vectors for DNA vaccine delivery.

scholarly journals Genetic dissection of centromere function.

1993 ◽  
Vol 13 (6) ◽  
pp. 3156-3166 ◽  
Author(s):  
I G Schulman ◽  
K Bloom
Keyword(s):  
Chromosome Segregation ◽  
Copy Number ◽  
Artificial Chromosome ◽  
Plasmid Copy Number ◽  
Genetic Dissection ◽  
Negative Effects ◽  
Plasmid Copy ◽  
Complex Cells ◽  
Copy Number Control

A system to detect a minimal function of Saccharomyces cerevisiae centromeres in vivo has been developed. Centromere DNA mutants have been examined and found to be active in a plasmid copy number control assay in the absence of segregation. The experiments allow the identification of a minimal centromere unit, CDE III, independently of its ability to mediate chromosome segregation. Centromere-mediated plasmid copy number control correlates with the ability of CDE III to assemble a DNA-protein complex. Cells forced to maintain excess copies of CDE III exhibit increased loss of a nonessential artificial chromosome. Thus, segregationally impaired centromeres can have negative effects in trans on chromosome segregation. The use of a plasmid copy number control assay has allowed assembly steps preceding chromosome segregation to be defined.

scholarly journals Genetic control of ColE1 plasmid stability that is independent of plasmid copy number regulation

2018 ◽  
Vol 65 (1) ◽  
pp. 179-192 ◽  
Author(s):  
Melissa S. Standley ◽  
Samuel Million-Weaver ◽  
David L. Alexander ◽  
Shuai Hu ◽  
Manel Camps
Keyword(s):  
Genetic Control ◽  
Copy Number ◽  
Plasmid Stability ◽  
Plasmid Copy Number ◽  
Plasmid Copy ◽  
Cole1 Plasmid ◽  
Copy Number Regulation

scholarly journals Genetic dissection of centromere function

1993 ◽  
Vol 13 (6) ◽  
pp. 3156-3166
Author(s):  
I G Schulman ◽  
K Bloom
Keyword(s):  
Chromosome Segregation ◽  
Copy Number ◽  
Artificial Chromosome ◽  
Plasmid Copy Number ◽  
Genetic Dissection ◽  
Negative Effects ◽  
Plasmid Copy ◽  
Complex Cells ◽  
Copy Number Control

A system to detect a minimal function of Saccharomyces cerevisiae centromeres in vivo has been developed. Centromere DNA mutants have been examined and found to be active in a plasmid copy number control assay in the absence of segregation. The experiments allow the identification of a minimal centromere unit, CDE III, independently of its ability to mediate chromosome segregation. Centromere-mediated plasmid copy number control correlates with the ability of CDE III to assemble a DNA-protein complex. Cells forced to maintain excess copies of CDE III exhibit increased loss of a nonessential artificial chromosome. Thus, segregationally impaired centromeres can have negative effects in trans on chromosome segregation. The use of a plasmid copy number control assay has allowed assembly steps preceding chromosome segregation to be defined.

scholarly journals Endogenous 2μ Plasmid Editing for Pathway Engineering in Saccharomyces cerevisiae

2021 ◽  
Vol 12 ◽  
Author(s):  
Bo-Xuan Zeng ◽  
Ming-Dong Yao ◽  
Wen-Hai Xiao ◽  
Yun-Zi Luo ◽  
Ying Wang ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Copy Number ◽  
Plasmid Stability ◽  
Essential Gene ◽  
Expression System ◽  
Plasmid Copy Number ◽  
Pathway Engineering ◽  
Plasmid Copy ◽  
Control Plasmid ◽  
Cell Variation

In Saccharomyces cerevisiae, conventional 2μ-plasmid based plasmid (pC2μ, such as pRS425) have been widely adopted in pathway engineering for multi-copy overexpression of key genes. However, the loss of partition and copy number control elements of yeast endogenous 2μ plasmid (pE2μ) brings the issues concerning plasmid stability and copy number of pC2μ, especially in long-term fermentation. In this study, we developed a method based on CRISPR/Cas9 to edit pE2μ and built the pE2μ multi-copy system by insertion of the target DNA element and elimination of the original pE2μ plasmid. The resulting plasmid pE2μRAF1 and pE2μREP2 demonstrated higher copy number and slower loss rate than a pC2μ control plasmid pRS425RK, when carrying the same target gene. Then, moving the essential gene TPI1 (encoding triose phosphate isomerase) from chromosome to pE2μRAF1 could increase the plasmid viability to nearly 100% and further increase the plasmid copy number by 73.95%. The expression using pE2μ multi-copy system demonstrated much smaller cell-to-cell variation comparing with pC2μ multi-copy system. With auxotrophic complementation of TPI1, the resulting plasmid pE2μRT could undergo cultivation of 90 generations under non-selective conditions without loss. Applying pE2μ multi-copy system for dihydroartemisinic acid (DHAA) biosynthesis, the production of DHAA was increased to 620.9 mg/L at shake-flask level in non-selective rich medium. This titer was 4.73-fold of the strain constructed based on pC2μ due to the more stable pE2μ plasmid system and with higher plasmid copy number. This study provides an improved expression system in yeast, and set a promising platform to construct biosynthesis pathway for valuable products.

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