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Plasmids 101: Simplify cloning with in vivo assembly

Posted by Guest Blogger on Oct 18, 2018 8:37:05 AM

This post was contributed by Jake Watson and Javier García-Nafría from the MRC Laboratory of Molecular Biology.

Plasmid cloning is an essential part of any molecular biology project, yet very often, it is also a bottleneck in the experimental process. The majority of current cloning techniques involve the assembly of a circular plasmid in vitro, before transforming it into E. coli for propagation. However, while not widely known, plasmid assembly can be achieved in vivo using a bacterial recombination pathway that is present even in common lab cloning strains.

This intrinsic bacterial recombination pathway, referred to as recA-independent recombination, joins together pieces of linear DNA through short homologous sequences at their termini, and likely functions as a bacterial DNA repair mechanism. The pathway is ubiquitous, with successful recombination reported in all laboratory E. coli strains tested so far.

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Topics: Plasmids 101, Plasmid Cloning

Plasmids 101: Codon usage bias

Posted by Tyler Ford on Sep 27, 2018 9:09:41 AM

A similar genetic code is used by most organisms on Earth, but different organisms have different preferences for the codons they use to encode specific amino acids. This is possible because there are 4 bases (A, T, C, and G) and 3 positions in each codon. There are therefore 64 possible codons but only 20 amino acids and 3 stop codons to encode leaving 41 codons unaccounted for. The result is redundancy; multiple codons encode single amino acids. Evolutionary constraints have molded which codons are used preferentially in which organisms - organisms have codon usage bias.

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Topics: Plasmids 101

Plasmids 101: 5 factors to help you choose the right cloning method

Posted by Michael G. Lemieux on Aug 21, 2018 8:31:59 AM

You’ve spent days and weeks thinking of an amazing project. You’ve written your protocols, designed your experiments, and prepared your reagents. You’re going to engineer the best thing since CRISPR; you are ready to clone! But...how?

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Topics: Plasmids 101, Plasmid Cloning

Plasmids 101: Protein Expression

Posted by Alyssa Cecchetelli on Jun 7, 2018 9:17:55 AM

The central dogma in molecular biology is DNA→RNA→Protein. To synthesize a particular protein DNA must first be transcribed into messenger RNA (mRNA). mRNA can then be translated at the ribosome into polypeptide chains that make up the primary structure of proteins. Most proteins are then modified via an array of post-translational modifications including protein folding, formation of disulfide bridges, glycosylation and acetylation to create functional, stable proteins. Protein expression refers to the second step of this process: the synthesis of proteins from mRNA and the addition of post-translational modifications

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Topics: Plasmids 101, Plasmid How To

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: Plasmids 101

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