This plasmid has 2 ORIGIN stories 🐣 BUT we only use 1 of them to REPLICATE & pass down our protein’s tale! So why does my pET Hector the Vector have 2 ORIGINS OF REPLICATION (ORI)s? 🤷‍♀️ Could he be a PHAGEMID?! 🤨 The bumbling biochemist is on the case…🕵️‍♀️

There’s a cool protein we want to study 😎 so we want to make lots of it, then purify it, & run experiments with it 🤓We’ve decided we’re going to try expressing it recombinantly in (harmless) bacteria 👉 i.e. we’re going to CLONE the genetic instructions for the protein into a small circular piece of DNA (PLASMID) ⭕️ that will serve as a vehicle (VECTOR)🚗 to take our gene into the cells & keep it there🔒 so the bacteria can make protein from it 🤗 This vector will remain separate from the bacteria’s own DNA & will replicate autonomously (it doesn’t have to wait for the bacteria to copy its own DNA in order to copy itself)

There’s more than just the gene inside the plasmid, but not “too much more” 👉 we want to keep it to the bare minimum 👉 it takes ⏰ & resources for the cell to copy the plasmid, so why have it waste time copying something you don’t need? 🤷‍♀️ Also, plasmids can only reach a certain size 👉 you can make more room for bigger genes by removing stuff you don’t need (& then different vectors can add in different bells & whistles to do things like tag your protein to make purification easier 🏷 or provide a “reporter” that fluoresces (lights up) when your protein’s made 🚨

BUT there’s some stuff that all plasmids need, including an ORIGIN OF REPLICATION (ORI), which serves as a start point for copying the plasmid’s DNA 🏁 And speaking of starts, let’s start by clarifying start sites to (hopefully) avoid confusion 🙃

In order to make protein from our gene, our gene (& only our gene) 1st has to be copied into RNA (DNA’s chemical cousin) 👉 this kind of DNA➡️RNA copying is called TRANSCRIPTION 👉 Its start sites are signaled by sequences called PROMOTERS & the copying’s carried out by proteins called RNA Polymerases (RNA Pol) 👍 BUT that’s NOT what we’re 💬 now!

Instead, the ORI is a sequence that signals the start site for REPLICATION 👉 rather than copying a single strand of a single gene from DNA to RNA, replication duplicates BOTH STRANDS of the ENTIRE PLASMID 👉 & the duplicate copy is in DNA form 👉 it’s an exact copy. 👍 This copying is carried out by DNA Polymerase (DNA Pol)(like we use in PCR) 🚂

All cells in all organisms have to be able to carry out such REPLICATION 👆or else they couldn’t pass down their genetic information 😬 It’s easy to visualize this with bacteria 💭 they reproduce just by splitting themselves in 2 😅 BUT if they don’t duplicate their DNA first, each of the 2 “daughter cells” would only get ½ genetic info & then their daughters would only get ½ of that, etc… so the cells would be missing genes to suit their needs! 😬 So they duplicate their DNA first 👍

Normally, cells only duplicate their DNA right before they’re going to divide (you don’t want all that extra DNA hanging around). When it’s time ⏰ DNA Pol binds ORI & (w/help) melts apart the 2 DNA strands & copies them 👉 To make this easier, the ORI’s usually AT rich (the DNA letters A & T form weaker interstrand bonds than G & C, making them easier to unzip) 👍

In our recombinant expression scenario, we don’t *just* need to copy the bacterial DNA, we ALSO need to copy our plasmid DNA so that we didn’t put in all that hard work getting it in there only for there not to be enough to get passed down! So we need our plasmid to have its own ORI. 👉 Usually, this is DIFFERENT from host cell’s ORI so the plasmid doesn’t have to wait for the bacteria to decide to divide before it copies itself 👉 allows you to have lots of copies of your plasmid inside each cell so you can hopefully make lots of protein🤞It still uses the host’s copying machinery (including the DNA Pol) but it recruits it differently. 👍

I got confused when I 👀 at this pET vector (pET stands for plasmid Expression vector under T7 control & it’s one of our go-to tools for RECOMBINANT PROTEIN EXPRESSION in BACTERIA) bc it has TWO ORIs 👉 a pBR322 ORI (the 1 we want) & something labeled “f1 ORI” 🤯 What gives? 🤷‍♀️

Better yet, what does *it* give? The f1 ORI will only give you single-stranded DNA 👉 it’s the origin from the f1 filamentous phage 👉 a phage (bacteriophage) is a virus that infects bacteria & plasmids like this that have BOTH bacterial & phage ORIs are called PHAGEMIDs 👍

F1 phage gets bacteria it infects to make ssDNA that it stuffs into viral packages & sends out to infect other cells. It makes a lot of single-stranded copies in the process, so in the past, scientists took advantage of this a lot to get ssDNA they could use for sequencing the plasmid to check what’s in there 🧐 BUT now we have PCR so we don’t need this

But it can still be useful for some things like mutagenesis & it doesn’t take up too much room, so it’s probably more work to take it out than it’s worth 🤷‍♀️ Especially since the cells can’t even use it unless you infect the host cell w/a “helper phage” like VCSM13 or M12K07 that provides the viral components needed to replicate it, package it up, & ship it out as phages.

So with the help of helper phages, Hector does double duty 👉 uses regular ORI to make more ds-plasmid & f1 ORI to make ss versions that are secreted as phage particles 👍

more on pET vectors:

more on recombinant expression:

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