You too can be a VECTOR elector! For PLASMID-term elections turn to antibiotic-based selection. Proteins are like molecular workers made up of amino acid letters that go around doing cool stuff in your cells. But your cells have to make them first – and I like to compare this protein-making to baking! And PLASMID VECTORS allow us to share recipes with (harmless) bacteria to get them to “bake our cakes” for us. Plasmids are little circular pieces of DNA that act as “extra cookbooks” we can sneak into bacteria to have the bacteria make more copies of the recipe and/or the finished good. But what to look for in a good vector? 

Cells are like bakeries & if you think of proteins as baked goods like cookies and cakes, their recipes are written in DNA in the form of GENES. When a cell wants to do the protein equivalent of baking a cake it first makes messenger RNA (mRNA) copies of the recipe (a process called TRANSCRIPTION) and sends the recipe copy to the chefs (ribosomes), which travel along the recipe, reading the instructions and adding the ingredients (amino acids) accordingly. More here:  http://bit.ly/300UPUk

Lots of genes are collected into “cookbooks” called CHROMOSOMES which have gene after gene after gene after… all wound up tight and stuffed into a membrane-bound compartment of cells called the NUCLEUS. In EUKARYOTES that is… Organisms like humans, plants, & fungi have such membrane-bound “rooms” within their cells BUT PROKARYOTES (like bacteria) don’t. They still have DNA & they still hold it in chromosomes (often just 1) BUT they don’t have a dedicated compartment for it.

If you think of cells as cookbook libraries/bakeries nuclei are kinda like “reference sections” of a library – permanent copies of recipes are stored in here but they can’t be “checked out” but the chefs aren’t allowed in. So copies have to be made. But in prokaryotes like bacteria the recipes (originals and copies) all get to hang out together with the chefs in 1 big compartment. And if you can sneak an extra recipe in there, they’ll make that too (hopefully…). They can do this because, in addition to their chromosomal DNA, which contains all the recipes they need to make more identical copies of themselves & carry out all their required functions, bacteria can contain “extra” pieces of DNA in the form of PLASMIDS.

PLASMIDS are “extrachromosomal” (not part of the chromosomes), circular pieces of DNA. Similarly to chromosomes, they’re double-stranded, which means they can easily be “unzipped” & copied (thanks to the 1:1 base-pair-ability of DNA & RNA each strand can serve as a template for the other strand) Each time the cell divides to make more cells it has to copy its chromosome so that the new “daughter cells” get a copy. Plasmids use the host’s Zerox machine (DNA polymerase), BUT they don’t have to wait for the host to divide to copy themselves. can make LOTS of copies of themselves. When the cell does divide, these copies will get split between the daughter cells, so they’ll inherit the plasmid as well.

In addition to such “vertical gene transfer” (parent cell to daughter cells), bacteria can also share plasmids w/other “adults” through “horizontal gene transfer (HGT)” in a process called CONJUGATION, 1 cell can take in a plasmid from another cell. Why would it want to? Plasmids can contain useful things like antibiotic resistance genes. So a bacterium can share a plasmid w/its buddies & help them survive.

In lab we take advantage of plasmids to put genes we want to study into bacteria to have them make more of that gene &/or protein it codes for. We can “repurpose” plasmids & customize them to our liking. We take out anything unnecessary & keep the important parts & add in anything we need. It’s like taking a little cookbook and swapping out a recipe. Now we have that recipe in a form that can get “donated” to a bacterial cookbook library!

That is, the plasmid can now act as a VECTOR – a vehicle for taking things we want into cells. Just like mosquitos can be vectors for malaria, plasmids can be vectors for genes we want to study. BUT just like not all mosquitos are vectors, not all plasmids are vectors. I mean, they’re always carrying *something*, but we usually only call them vectors when we they’re carrying the thing we want them to carry. 

We’re the plasmid cookbook editors, so we get the design them to carry the somethings we want. So what *do* we want in our vector? There are a few main things we’ll need.

ORIGIN OF REPLICATION (ORI): this serves as an “unzipping start site”. When the plasmid copies itself, it starts here. The plasmid will replicate using the host cell’s machinery, but it usually has a different ORI than the host chromosomes so you can make multiple plasmid copies w/o waiting for host cell to divide.

different ORIs can give you different “copy numbers” which refers to how many copies of it there are on average in a cell. if you want to AMPLIFY a gene (make lots of DNA copies of it) you want a high copy #  BUT if you want to EXPRESS a gene (turn it into product), too high copy number can actually be detrimental because you want the cells to use their resources to make RNA & protein, NOT more DNA! (And speaking of conserving resources, this is one reason to take out unneeded recipes. Another reason you might want a low copy number is if your protein’s toxic to the cell – asking each cell to make a lot of it can quickly kill them, so you’re better off growing more cells but having each cell make less. more here: http://bit.ly/2RHtuSg

RESTRICTION SITES: These are specific DNA sequences (usually just 8-ish letters long that act like “cut along the dotted line” “dotted lines” for picky scissors -> they act as cut sites for different proteins called RESTRICTION ENZYMES (sequence-dependent endonucleases). If you add the specific corresponding restriction enzyme it’ll cut there (& only there), openning up the plasmid to let you put in the gene you want. more here: http://bit.ly/2sQaiYw

often plasmids are engineered to have a MULTIPLE CLONING SITE (MCS), which is a short stretch of DNA wi/cut sites for multiple enzymes so you can take your pick depending on what you want to cut your gene with (you want matching “sticky ends”) In addition to these “cut & paste” methods for getting your insert in there, you can use “copy & staple” methods like SLIC, which you can learn more about here: http://bit.ly/2Tkg5B1

SELECTION MARKER: usually an antibiotic resistance gene, this marker ensures only bacteria containing your plasmid can survive so you don’t waste your resources growing bacteria w/o your gene – e.g. if plasmid has an ampicillin resistance gene, you can grow bacteria (hopefully w/plasmid) on food spiked w/Amp & it’ll “weed out” any bacteria that don’t have it. More on such antibacterial selection here: http://bit.ly/2tcW4ky

PROMOTER: If you want gene to be expressed (turned into RNA (transcribed) &/or protein (translation))(i.e. you want an EXPRESSION VECTOR) you need to provide a “launch pad” for the transcriber (RNA polymerase) to start copying from. Promoters are “host specific” – you might need a differerent promoter for different host cells (e.g. there are plasmids specialized to be put into different hosts, even mammalian ones so we can do experiments in “tissues in a dish”)

As we talked about, we want to be “frugal” so the cells focus on our protein (and because it’s harder to sneak big cookbooks in… But we can add lots of extra “bells & whistles” when needed. like “reporters” that give a signal (such as production of a fluorescent protein ) that a gene’s being expressed or “tags” that tail a protein to make it easier to pull out & purify. more here: http://bit.ly/2XBu9eI

INSERTION: Speaking of using plasmids to make proteins… instead of putting in the “genomic” version of gene (gDNA) letter-for-letter like it’s written in our chromosomes, we put in complementary DNA (cDNA) which is an edited version.  It’s complementary to processed messenger RNA (mRNA) so it doesn’t have “margin notes” called introns that bacterial cells can’t remove for us. more http://bit.ly/2AsVdjG 

If we want to study a gene, thankfully we often don’t have to start from scratch because when scientists make plasmids they often deposit samples of it in “plasmid repositories” so other researchers can use them as well & some companies specialize in providing plasmids.

⚠️ BUT ALWAYS SEQUENCE YOUR PLASMIDS to double-check that the gene they say’s there’s really there, It’s not that scientists or companies are trying to sabotage you, but they’re dealing w/LOTS of plasmids, & accidental mix-ups occasionally occur (learned this hard way… 😩)

Some notes on terminology: When we stick a new gene into a plasmid, we’re “recombining” DNA so we call product RECOMBINANT DNA & since we’re making more copies, we’re CLONING the gene. So I do a LOT of cloning, but I’m cloning INDIVIDUAL GENES, not entire individuals! Often, when you can get cDNA through one of those repositories it comes in a plasmid that doesn’t have everything you need plasmid cDNA comes in doesn’t have everything u need. So you can add in more stuff or cut it out of one plasmid & put it into another. We call this SUBCLONING. 

more on topics mentioned (& others) #365DaysOfScience All (with topics listed) 👉 http://bit.ly/2OllAB0

Leave a Reply

Your email address will not be published.