Plasmid Overexpression
Plasmid Overexpression - Related Videos
08:57
High-throughput Yeast Plasmid Overexpression Screen
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Cited by 12 •
2011
Here we describe a plasmid overexpression screen in Saccharomyces cerevisiae, using an arrayed plasmid library and a high-throughput yeast transformation protocol with a liquid handling robot.
08:16
Plasmid Purification
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2023
Plasmid purification is a technique used to isolate and purify plasmid DNA from genomic DNA, proteins, ribosomes, and the bacterial cell wall. A plasmid is a small, circular, double-stranded DNA that is used as a carrier of specific DNA molecules. When introduced into a host organism via transformation, a plasmid will be replicated, creating numerous copies of the DNA fragment under study. In this video, a step-by-step generalized procedure is described for how to perform plasmid purification.
01:28
Plasmids
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2025
Plasmids are extrachromosomal DNA molecules found in bacteria, archaea, and some eukaryotic microbes like yeast. These small, circular DNA structures typically contain fewer than 30 genes, although some may exist linearly. Plasmids vary in their number within a cell, known as copy number. Single-copy plasmids are present in one copy per cell and multi-copy plasmids are present in multiple copies, reaching over 100 copies per cell.Plasmids usually replicate independently of the chromosomal DNA...
07:11
Homemade Site Directed Mutagenesis of Whole Plasmids
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Cited by 91 •
2009
Site directed mutagenesis of whole plasmids is a simple way to create slightly different variations of an original plasmid. Here we demonstrate an easy and cost effective way to introduce base substitutions into a plasmid using standard reagents.
05:16
Detecting Histone Modifications in Yeast Cells with Neurodegenerative Protein Overexpression
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2025
Source: Bennett, S. A., et al., Characterizing Histone Post-translational Modification Alterations in Yeast Neurodegenerative Proteinopathy Models. J. Vis. Exp. (2019).This video demonstrates the procedure of assessing histone modifications in yeast cells overexpressing neurodegenerative proteins. The steps include cell lysis, centrifugation, electrophoresis, membrane transfer, and antibody-based detection of modified histones.
FAQs
The promoter controls how strongly and when the inserted gene is transcribed. A strong promoter can support high messenger RNA production, while regulation allows researchers to control expression rather than relying entirely on constitutive activity. This control matters because the resulting protein output depends partly on promoter behavior and on the induction conditions used in the experiment.
Plasmid copy number changes how many copies of the recombinant template are present, while host biology affects how efficiently those cells transcribe the gene and translate its messenger RNA. Consequently, the same construct can produce different protein amounts in different cellular systems. These variables must be considered when comparing expression results or interpreting unusually high or low production.
Induction conditions determine how the regulatable promoter is activated and therefore influence the amount of protein produced. Because output is not controlled by the plasmid sequence alone, researchers treat induction as an experimental variable when evaluating expression. Keeping this factor in view helps explain differences between preparations and supports more meaningful comparisons of protein production.
A typical workflow uses a recombinant plasmid carrying the gene, introduces it into appropriate host cells by transformation, and then permits transcription and translation from the inserted sequence. Researchers subsequently examine the resulting protein for functional or production-related goals. This sequence connects the genetic construct with the protein-level outcome being studied.
Applications extend beyond simply increasing protein abundance. Researchers use the method to investigate what a gene does, analyze the activity of its protein product, produce recombinant proteins, and model cellular pathways. These uses make the approach valuable both for testing gene function and for examining how a chosen protein contributes to a broader biological process.
Bacteria, yeast, and cultured mammalian cells are identified as host systems for this approach. Their differing host biology can influence transcription, translation, and the final protein yield, so the cellular context is part of experimental interpretation. Choosing among these systems therefore connects the overexpression experiment to the biological setting in which the protein is being examined.