Plasmids designed to express genes in a given host cell type are generally broken down into two broad categories, prokaryotic or eukaryotic, based on the functional elements they contain. Plasmid DNA in both prokaryotic and eukaryotic systems must be transcribed into RNA, which occurs in three phases: initiation, elongation, and termination. In a previous post we discussed the promoter's role in the initiation step of gene transcription; today we'll provide an overview on how transcription stops, or termination. Read on to learn more!
Have you ever found yourself bamboozled by all of the different kinds of information on our plasmid pages? Well, to help make the most of these pages, we've written this post to guide you through them and make the best use of all the information provided by your colleagues.
Plasmids utilize their host cell's replication machinery in order to replicate. As described in our previous Origin of Replication post, DNA replication is initiated at the ORI and may be synchronized with the replication of the host cell's chromosomal DNA or may be independent of the host's cell cycle.
Plasmids are said to be under stringent control of replication when they are dependent on the presence of initiation proteins synthesized by the host cell in order to start their own replication. In general, these types of plasmids tend to be low copy number. Conversely, plasmids that can initiate DNA replication independently of the host's initiation proteins are said to be under relaxed control, as they only require the host's replication machinery for elongation and termination. These types of plasmids tend to be high copy number.
First described in the 1980s, protein tags are now one of the most useful items in a scientist’s toolbox. As we’ve covered in Plasmids 101, tags can help you determine localization of a protein of interest, purify it, or determine its expression level without the need for a custom antibody. There is one major caveat - a tag may interfere with protein localization and/or function, so each tagged protein must be tested carefully to ensure it retains the attributes of the native protein. Since this process takes a lot of time and energy, Martí Aldea and collaborators have created a set of “innocuous tags” (inntags) less likely to alter a protein’s properties.
This post is part of our ongoing Plasmids 101 series. Plasmids 101 will provide you with an overview of general molecular biology knowledge and techniques. If you are interested in reading more, you can find the rest of the Plasmids 101 posts here.
Now that we have covered antibiotic selection here at Plasmids 101, we can talk about an even more specific method of screening your cloning reaction. Being able to select for colonies that contain your plasmid is a great start when cloning, but how about being able to choose those that contain plasmid with an insert? Blue-white selection is a widely used method to do just that!