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Plasmids 101: Origin of Replication

Posted by Kendall Morgan on Nov 11, 2020 9:23:38 AM

Originally published Feb 6, 2014 and last updated Nov 10, 2020.

Now that we know all about antibiotic resistance genes, let’s consider another basic element of any plasmid: the origin of replication/replicon. The replicon is comprised of the origin of replication (ori) and all of its control elements. The ori is the place where DNA replication begins, enabling a plasmid to reproduce itself as it must to survive within cells.

The replicons of plasmids are generally different from the those used to replicate the host's chromosomal DNA, but they still rely on the host machinery to make additional copies. ori sequences are generally high in As and Ts. Why, you ask? Well, A-T base pairs are held together with two hydrogen bonds not three as G-C pairs are. As a result, stretches of DNA that are rich in A-T pairs can be separated more readily at lower temperatures and allows the replication machinery room to come in and get busy making copies.

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Topics: Plasmid Elements, Plasmids 101, Plasmids

Bacterial Toxin-antitoxin Systems as Molecular Biology Tools

Posted by Jennifer Tsang on Nov 1, 2018 8:35:58 AM

Members of the bacterial world produce an assortment toxins to claim territory or kill competing microorganisms, but did you know bacteria also produce substances toxic to themselves?

What are toxin-antitoxin systems?

These toxic substances are part of toxin-antitoxin systems that are widely present in bacteria. They consist of a toxin which can affect a variety of cellular processes and an antitoxin that suppresses the toxin’s activity. The key to these systems is that the toxin is stable while the antitoxin is unstable, meaning that the cells must continually produce antitoxin to avoid cell death.

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Topics: Plasmid Elements, Plasmids

Stabilized Bacterial Promoters: Constant Gene Expression at any Copy Number

Posted by Jennifer Tsang on Sep 4, 2018 8:53:28 AM

Researchers express genes of interest from plasmids in order to study gene function or to engineer cells for specific purposes. Unfortunately, plasmid copy numbers vary within cell populations and over time resulting in variable gene expression that can impact observed phenotypes. Factors such as the growth medium, growth temperature, and growth rate can all impact plasmid copy number in a cell.

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Topics: Plasmid Elements, Other Plasmid Tools, Plasmids

Plasmids 101: Repressible Promoters

Posted by Mary Gearing on May 10, 2018 9:15:54 AM

Promoters control the binding of RNA polymerase and transcription factors. Since the promoter region drives transcription of a target gene, it therefore determines the timing of gene expression and largely defines the amount of recombinant protein that will be produced. Many common promoters like T7, CMV, EF1A, and SV40, are always active and thus referred to as constitutive promoters. Others are only active under specific circumstances. In a previous post, we discussed inducible promoters, which can be switched from an OFF to an ON state, and how you might use these in your research. Today, we’ll look at repressible promoters, which can be switched from an ON to an OFF state, as well as repressible binary systems commonly used in Drosophila.

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Topics: Plasmid Elements, Plasmids 101, Plasmids

Plasmids 101: Inducible Promoters

Posted by Mary Gearing on Jan 18, 2018 9:34:59 AM

Promoters control the binding of RNA polymerase and transcription factors. Since the promoter region drives transcription of a target gene, it therefore determines the timing of gene expression and largely defines the amount of recombinant protein that will be produced. Many common promoters. like CMV, EF1A, and SV40 promoters, are always active and thus referred to as constitutive promoters. Others are only active under specific circumstances. In this post, we’ll discuss inducible promoters, which can be switched from an OFF to an ON state, and how you might use these in your research. When you're done with this post, check out our follow up post on repressible promoters.

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Topics: Plasmid Elements, Plasmids 101, Plasmids

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