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Plasmids 101: Green Fluorescent Protein (GFP)

Posted by Marcy Patrick on May 15, 2014 11:33:00 AM

Bioluminescence and fluorescence from proteins such as Green Fluorescent Protein (GFP) has likely existed in creatures such as jellyfish for millions of years; however, it took until the 1960s for scientists to begin to study GFP and deduce its biochemical properties. Now GFP and its fluorescent derivatives are a staple in the lab. GFP is used in research across a vast array of biological disciplines and scientists employ GFP for a wide number of functions, including: tagging genes for elucidating their expression or localization profiles, acting as a biosensor or cell marker, studying protein-protein interactions, visualizing promoter activity, and much more.

Read on to learn more about GFP, how scientists have evolved this versatile protein to suit their experimental needs, and some of the common applications in the lab.

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Topics: Plasmid Elements, Plasmids 101, Fluorescent Proteins

Your Lentiviral Plasmid FAQs Answered

Posted by Kendall Morgan on Apr 23, 2014 9:08:00 AM

Lentiviruses are useful and efficient tools to introduce your gene of interest into cells. Unlike gamma-retroviruses that can only infect dividing cells, lentiviruses can infect dividing and non-dividing cells. 

Addgene has an extensive collection of lentiviral plasmids created for a variety of applications including cDNA expression, shRNA-mediated knockdown, Tet and Cre-regulated expression, CRISPR genome editing, and more. Not surprisingly, we receive many questions from scientists all over the world looking for some additional information or clarification on these vectors. Read on to find the answers to our most frequently asked lentiviral questions.

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Topics: Plasmid How To, Plasmid Elements, Lab Tips, Viral Vectors

Plasmids 101: The Promoter Region – Let's Go!

Posted by Kendall Morgan on Apr 3, 2014 4:05:00 PM

Thus far in our Plasmids 101 series we've worked our way through the plasmid map: antibiotic resistance, origin of replication, and so on. Up to this point we can replicate our plasmid and make sure cells maintain it; the next step is getting the plasmid to express our gene of interest. Enter the promoter-- the element responsible for initiating the transcription of your insert into RNA.

In practice, the term "promoter" describes the combination of the promoter (RNA polymerase binding site) and operators (response elements). Promoters are about 100 to 1000 base pairs long and found upstream of their target genes. The sequence of the promoter region controls the binding of the RNA polymerase and transcription factors, therefore promoters play a large role in determining where and when your gene of interest will be expressed. 

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Topics: Plasmid Elements, Plasmids 101

Plasmids 101: Yeast Vectors

Posted by Marcy Patrick on Feb 25, 2014 2:11:00 PM

In our first few Plasmids 101 posts, we focused mainly on the elements required for plasmid maintenence within an E. coli cell, but vectors can be widely utilized across many different cell types and each one requires different elements for vector propogation. This post, along with a future companion post on mammalian vectors, will catch you up on the core replication and resistance features of yeast vectors and explain how they differ from the bacterial elements previously described.

Why Do Scientists Use Yeast Vectors?

Yeast are eukaryotes and thus contain complex internal cell structures similar to those of plants and animals. Unlike bacteria, yeast can post-translationally modify proteins yet they still share many of the same technical advantages that come with working with prokaryotes. This includes but is not limited to: rapid growth, ease of replica plating and mutant isolation, a well-defined genetic system, and a highly versatile DNA transformation system.

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Topics: Plasmid How To, Plasmid Elements, Lab Tips, Plasmids 101

Plasmids 101: Origin of Replication

Posted by Kendall Morgan on Feb 6, 2014 10:25:00 AM

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 melt more readily at lower temperatures. When DNA melts, it gives the replication machinery room to come in and get busy making copies.

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Topics: Plasmid How To, Plasmid Elements, Plasmids 101

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