A new combinatorial megaplasmid library assembly method designed to screen for minimal pathways by using SCRaMbLE

Human proteins expressed in yeast are common to enhance protein production while the expression of functional human pathways remain challenging. Here, we propose a simple and economical high-throughput gene assembly method to create a yeast megaplasmid library from human cDNA to screen for minimal h...

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Bibliographic Details
Main Authors: Yap, Wei Sheng, Thibault, Guillaume
Other Authors: School of Biological Sciences
Format: Article
Language:English
Published: 2023
Subjects:
Online Access:https://hdl.handle.net/10356/165616
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Institution: Nanyang Technological University
Language: English
Description
Summary:Human proteins expressed in yeast are common to enhance protein production while the expression of functional human pathways remain challenging. Here, we propose a simple and economical high-throughput gene assembly method to create a yeast megaplasmid library from human cDNA to screen for minimal human functional pathways. We introduced artificial promoters followed by symmetric loxP sites into the megaplasmids using Golden Gate assembly coupled with streptavidin-bead-based purification. The isolated high molecular weight, randomly assembled cDNA megaplasmid library may be useful for high-throughput directed evolution experiments and may be adapted for use in other model organisms.