scholarly journals Yeast transformation efficiency is enhanced by TORC 1‐ and eisosome‐dependent signaling

2018 ◽  
Vol 8 (5) ◽  
pp. e00730 ◽  
Author(s):  
Sheng‐Chun Yu ◽  
Florian Kuemmel ◽  
Maria‐Nefeli Skoufou‐Papoutsaki ◽  
Pietro D. Spanu
Keyword(s):  
Transformation Efficiency ◽  
Yeast Transformation

scholarly journals Nutrient supplements boost yeast transformation efficiency

2016 ◽  
Vol 6 (1) ◽  
Author(s):  
Sheng-Chun Yu ◽  
Alexander Dawson ◽  
Alyssa C. Henderson ◽  
Eloise J. Lockyer ◽  
Emily Read ◽  
...  
Keyword(s):  
Transformation Efficiency ◽  
Yeast Transformation ◽  
Nutrient Supplements

626 Improving the yeast transformation efficiency for yeast display in antibody development

2020 ◽  
Vol 8 (Suppl 3) ◽  
pp. A662-A662
Author(s):  
Jian Chen ◽  
George Sun
Keyword(s):  
Development Process ◽  
Transformation Efficiency ◽  
Scaling Up ◽  
Affinity Maturation ◽  
Yeast Transformation ◽  
Yeast Display ◽  
Library Size ◽  
New Device ◽  
Display Technology ◽  
Single Reaction

BackgroundIn the therapeutic antibody development process, the yeast display technology which expresses a large library of antibodies is very useful for increasing the affinity of a lead antibody. Ideally, a yeast library should exceed the size of 10E10 to 10E11 to get close to the real affinity maturation process. However, due to low transformation efficiency with yeast, it requires trememdous scaling-up efforts to simply reach the 10E9 library size.MethodsTo address the transformation problem, we developed a new electroporation device that applies a high voltage on a sealed electroporation tube containing the yeast and plasmids in a low conductance buffer.ResultsThe new device is arcing free due to the sealed design and each single reaction could generate 10E8 library size, far exceeding the 10E6 size that was previously reported in a single reaction.ConclusionsWith the improved transformation efficiency, it becomes very straightforward to reach the currently difficult size of 10E9. Further more, it is possible to reach the 10E10 to 10E11 library size with reaction scaling-up. Our new method could be very useful for the field of antibody development.

scholarly journals Yeast transformation efficiency is enhanced by TORC1- and eisosome-dependent signalling

2018 ◽  
Author(s):  
Sheng-Chun Yu ◽  
Florian Kuemmel ◽  
Maria-Nefeli Skoufou-Papoutsaki ◽  
Pietro D Spanu
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Amino Acids ◽  
Gene Therapy ◽  
Molecular Mechanisms ◽  
Transformation Efficiency ◽  
Fold Increase ◽  
Dna Uptake ◽  
Yeast Transformation ◽  
Transformation Medium ◽  
Membrane Compartment

ABSTRACTTransformation of baker’s yeast (Saccharomyces cerevisiae) plays a key role in several experimental techniques, yet the molecular mechanisms underpinning transformation are still unclear. Addition of amino acids to the growth and transformation medium increases transformation efficiency. Here, we show that target of rapamycin complex 1 (TORC1) activated by amino acids enhances transformation via ubiquitin-mediated endocytosis. We created mutants of the TORC1 pathway, α-arrestins, and eisosome-related genes. Our results demonstrate that the TORC1-Npr1-Art1/Rsp5 pathway regulates yeast transformation. Based on our previous study, activation of this pathway results in a 13-fold increase in transformation efficiency, or greater. Additionally, we suggest DNA is taken up by domains at the membrane compartment of Can1 (MCC) in the plasma membrane formed by eisosomes. Yeast studies on transformation could be used as a platform to understand the mechanism of DNA uptake in mammalian systems, which is clinically relevant to optimise gene therapy.

scholarly journals Free fatty acids promote transformation efficiency of yeast

2019 ◽  
Vol 19 (7) ◽  
Author(s):  
Xingpeng Duan ◽  
Xiaojing Ma ◽  
Shengying Li ◽  
Yongjin J Zhou
Keyword(s):  
Transformation Efficiency ◽  
Culture Media ◽  
Cell Factory ◽  
Competent Cell ◽  
Cell Preparation ◽  
Yeast Transformation ◽  
High Transformation Efficiency ◽  
Metabolic Analysis ◽  
High Transformation ◽  
Large Plasmids

ABSTRACT High transformation efficiency is essential in genetic engineering for functional metabolic analysis and cell factory construction, in particular in construction of long biosynthetic pathways with multiple genes. Here, we found that free fatty acid (FFA)-overproducing strain showed higher transformation efficiency in Saccharomyces cerevisiae. We then verified that external supplementation of FFAs, to the culture media for competent cell preparation, improved yeast transformation efficiency significantly. Among all tested FFAs, 0.5 g/L C16:0 FFA worked best on promoting transformation of S. cerevisiae and Komagataella phaffii (previously named as Pichia pastoris). Furthermore, C16:0 FFA improved the assembly efficiency of multiple DNA fragments into large plasmids and genome by 100%, which will facilitate the construction and optimization of multigene-containing long pathways.

scholarly journals Optimizing Transformation Frequency of Cryptococcus neoformans and Cryptococcus gattii Using Agrobacterium tumefaciens

2021 ◽  
Vol 7 (7) ◽  
pp. 520
Author(s):  
Jianmin Fu ◽  
Nohelli E. Brockman ◽  
Brian L. Wickes
Keyword(s):  
Agrobacterium Tumefaciens ◽  
Yeast Cell ◽  
Transformation Efficiency ◽  
Transformation Frequency ◽  
Yeast Cells ◽  
Agar Concentration ◽  
Genetic Tool ◽  
Number Of Factors ◽  
Cryptococcus Spp ◽  
Transformation Systems

The transformation of Cryptococcus spp. by Agrobacterium tumefaciens has proven to be a useful genetic tool. A number of factors affect transformation frequency. These factors include acetosyringone concentration, bacterial cell to yeast cell ratio, cell wall damage, and agar concentration. Agar concentration was found to have a significant effect on the transformant number as transformants increased with agar concentration across all four serotypes. When infection time points were tested, higher agar concentrations were found to result in an earlier transfer of the Ti-plasmid to the yeast cell, with the earliest transformant appearing two h after A. tumefaciens contact with yeast cells. These results demonstrate that A. tumefaciens transformation efficiency can be affected by a variety of factors and continued investigation of these factors can lead to improvements in specific A. tumefaciens/fungus transformation systems.

scholarly journals WUSCHEL Overexpression Promotes Callogenesis and Somatic Embryogenesis in Medicago truncatula Gaertn

Plants ◽  
2021 ◽  
Vol 10 (4) ◽  
pp. 715
Author(s):  
Aline Kadri ◽  
Ghislaine Grenier De March ◽  
François Guerineau ◽  
Viviane Cosson ◽  
Pascal Ratet
Keyword(s):  
Somatic Embryogenesis ◽  
Medicago Truncatula ◽  
Transformation Efficiency ◽  
Genetic Manipulation ◽  
Leaf Explants ◽  
Promising Tool ◽  
Limiting Step ◽  
Wus Gene ◽  
Multiple Signaling ◽  
Regeneration Systems

The induction of plant somatic embryogenesis is often a limiting step for plant multiplication and genetic manipulation in numerous crops. It depends on multiple signaling developmental processes involving phytohormones and the induction of specific genes. The WUSCHEL gene (WUS) is required for the production of plant embryogenic stem cells. To explore a different approach to induce somatic embryogenesis, we have investigated the effect of the heterologous ArabidopsisWUS gene overexpression under the control of the jasmonate responsive vsp1 promoter on the morphogenic responses of Medicago truncatula explants. WUS expression in leaf explants increased callogenesis and embryogenesis in the absence of growth regulators. Similarly, WUS expression enhanced the embryogenic potential of hairy root fragments. The WUS gene represents thus a promising tool to develop plant growth regulator-free regeneration systems or to improve regeneration and transformation efficiency in recalcitrant crops.

scholarly journals Identification of highly transformable wheat genotypes for mass production of fertile transgenic plants

Genome ◽  
2002 ◽  
Vol 45 (2) ◽  
pp. 421-430 ◽  
Author(s):  
A Pellegrineschi ◽  
L M Noguera ◽  
B Skovmand ◽  
R M Brito ◽  
L Velazquez ◽  
...  
Keyword(s):  
Transformation Efficiency ◽  
Transformation Process ◽  
Plant Genotype ◽  
Donor Plant ◽  
Genotype Effect ◽  
Factors Influencing ◽  
Number Of Factors ◽  
Selection Medium ◽  
Callus Regeneration ◽  
Transformation Systems

The efficiency of wheat biolistic transformation systems strongly depends on the bombardment parameters, the condition of the donor plant, and the plant genotype chosen for the transformation process. This paper analyzes the transformation efficiency of the 129 wheat sister lines generically called 'Bobwhite', originally obtained from the cross 'Aurora'//'Kalyan'/'Bluebird 3'/'Woodpecker'. A number of factors influencing the transformation were examined, such as the ability to produce embryogenic callus, regeneration in selection medium, and overall transformation performance. Of the 129 genotypes evaluated, eight demonstrated transformation efficiencies above 60% (60 independent transgenic events per 100 immature embryos bombarded). Among the eight genotypes identified, we studied agronomic characteristics such as earliness to identify the most adaptable line(s) for different lab conditions. 'Bobwhite' SH 98 26 was identified as a super-transformable wheat line.Key words: wheat transformation, 'Bobwhite', genotype effect.

One way, but diverse methods for fungi and yeast transformation

2014 ◽  
Vol 11 (2) ◽  
pp. 204-205 ◽  
Author(s):  
Andrés Cruz Hernández ◽  
Jaime Ángeles Ángeles ◽  
Juan Campos-Guillén
Keyword(s):  
Yeast Transformation
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