If you want cells to make a protein for you, you’ll need to fill out a biochemical “order form” – but make sure you enter the correct genetic password! Did you get it right? How do I know if my molecular cloning went ok? I take different steps to check along the way!We can use circular pieces of DNA called plasmids as vectors (carriers) for the genetic information for making specific proteins we want to study. In a way these are are like biochemical “order forms” that tell the cells what we want to make.
We use MOLECULAR CLONING to “fill out the order form” & we need to make sure we get it all right. There are several ways we can get “hints” about whether we successfully cloned a gene of interest into a plasmid, BUT for definitive proof, we need SEQUENCING PRIMERS to help us “hack in” & check
In MOLECULAR CLONING we take a gene from one place and stick it into a smaller, circular, piece of DNA called a PLASMID VECTOR that has things to make it easy to work with (like being able to make lots of copies of itself and having an antibiotic resistance gene that will come in handy) & you stick it in (harmless) bacterial cells to make lots of copies of it for you. If you’re a protein person like me and you want the protein product from that gene not just the gene, you can then take your clone and put it into cells that will make protein from it.
Usually we do the cloning part in 1 type of bacteria that’s really good at making lots of copies of the gene because it’s “high copy number” meaning each cell can hold lots of copies of those “order forms” (I use DH5α for this). Then we break those cells open & take out that DNA, purify it, & check that it’s the right thing before we put the plasmid into another type of host cell that’s really good at making protein (sometimes these cells are also bacteria, but a different strain, like BL21(DE3).
You can think of it kinda like your plasmid is an online order form you “submit” to cells you want to express a protein and the gene containing the specific instructions for that protein is a (really long) “password” you have to enter. Biologically, what needs to happen to make a protein is that the DNA (permanent copy) gets transcribed into a temporary copy in the form of messenger RNA (mRNA) and that that mRNA gets “read” as 3-RNA-letter (nucleotide) words called codons that spell 1 protein-letter (amino acid) that gets added.
So the sequence of our “password” will ultimately lead to the sequence of the protein – and If you enter the wrong password you won’t get the protein you want – you might get no protein or you could get a “defective” protein – so you want to make sure you get it right.
The conclusive proof that it’s the correct sequence comes from DNA SEQUENCING but there are several ways we can get reassurance along the way, so we don’t waste time & money on sequencing the wrong DNA! Let’s look at how we know we’re on the right track during the cloning process (filling out that order form with the right password…)
That moment of panic – Did you even submit the form? (is my plasmid in these cells?) As long as you’ve used antibiotic selection you can rest easy on this front. You use a plasmid that has an antibiotic resistance gene that the host cells will need to survive if you add that antibiotic to their food. So only cells containing the plasmid will grow. This is a form of SELECTION that automatically “rejects” any cells that don’t have a form. http://bit.ly/2tcW4ky
Phew! You submitted the form – but did you put in a password? antibiotic selection only tells you that the bacteria have your plasmid – does NOT tell you that the plasmid has your gene
You can set up your cloning so that you get an “error message” if you forget to put in a password (you know those messages telling you you have not completed all the required fields…). But it will only tell you if you’ve put “something” (ANYTHING) in – blue-white screening works by putting your gene into the plasmid in the middle of a gene that bacteria need to turn a chemical you add (X-gal) blue. So if your gene gets inserted the bacteria will be white not blue. http://bit.ly/2MxNPs2
But just like you might have “tricked” a form by putting random letters for your contact info (you thought you could SPAM me, hah!) anything could be put in there and it’d still pass this test. Unlike the antibiotic test above, which was a SELECTION, this blue-white screening is a SCREEN -> it lets you know there’s a problem, but KEEPS the problematic ones there for you to see & avoid.
So how can we know what’s in there? You know how some digital forms let you toggle between showing the password as you type and hiding it and just showing dots or asterisks? If you’re in the asterisky mode you can tell how many characters you’ve typed but not if there are any typos. We know what the password should be, so we know how long it should be so knowing how many characters we typed gives us a good hint.
The way we can do this biochemically is to use colony PCR or an analytical restriction enzyme digest to see the size of the region that should have your gene. More on how these work here: http://bit.ly/30Npa8o & http://bit.ly/2IpBFAz &
but the basic idea is that you either cut out (in the digest) or make lots of copies of (in colony PCR) the part of your plasmid that should contain your gene. Then you see how many & how big those pieces are (with agarose gel electrophoresis). If your gene is there the piece will be much bigger than if it’s not there. And while you can’t tell exactly how many DNA letters are there, you get an idea whether you’re in the right ballpark. BUT you still don’t know if there are any typos! (is the sequence correct?)
Time for the definitive proof that we typed the right password! (note: I don’t usually do the colony PCR or digest step unless I’m having problems (often not worth it)). The conclusive proof that it’s the correct sequence comes from DNA SEQUENCING – but unlike the type of sequencing that sequences “all” your DNA, we’re only interested in sequencing the specific region with our gene.
Using sequencing primers is similar in setup and concept to vector-specific colony pcr – use 1 primer that matches a sequence upstream of your gene and one downstream. These primers are short pieces of DNA that serve as start points for DNA polymerase to start copying the DNA by adding matching bases.
In colony PCR you have both primers in the same reaction – so one primer starts on one strand – goes until it runs out of steam – and the same thing with the other primer and the other strand. Then in the next PCR cycle those pieces become the new template. they start at the other primer site and go till they fall off the end – which is defined by where the primer that made the strand started. So now you’re making defined-length pieces bookended by the 2 primer locations. let DNA pol do this over and over, copying and copying the DNA between the 2 primers so you have enough to run on a gel and see how big the product is.
For the sequencing reaction, instead of focusing on making tons of copies, you focus on reading carefully – you read out the sequence as you add each base. Instead of adding both primers in the same reaction, it’s one at a time, so instead of making double-stranded (ds) copies of a defined region of DNA, you start making a copy of a single strand and you “stalk it” as it works
You put in fluorescently-labeled nucleotides (nucleic acids) so you can “watch them be added” – most methods use special dye-terminator nucleotide methods where the fluorescently-labeled letters are “defective” – they’re dideoxynuclotides (as opposed to the “normal” singly-oxygen-defficient deoxynuclotides (dNTPs). DNA has 1 less oxygen than RNA (at the 2’ position (“right leg” of the sugar) – and ddNTPs are also missing the 3’OH oxygen (“left leg”) so there’s nowhere for more nucleotides to be added after it – it thus acts as a chain terminator – and if it’s fluorescently-labeled (with different colors for the different letters) you can see what letter it ended in
You put in a mix of unlabeled, normal letters and labeled defective letters so you get pieces that all start at the same place (primer binding site) but stop at different letters ofter traveling different distances – you can run those pieces through capillary electrophoresis to separate them by size (like a really long, thin version of the agarose slab gels we often run) and you shine a laser at them as they travel so you can “read out” what letter the pieces end in then read out the sequence. more here: http://bit.ly/2koF5el
To make gene-in-plasmid checking easier, plasmids often contain “standard” sequencing primer sites flanking the gene insertion site. These match standard primers that the sequencing companies will often provide for free – just ship them your plasmid, tell them what to use & they’ll send you the sequences.
But you can also use your own sequencing primers. I have primers that match the regions of the plasmids I commonly use right before and after where the gene goes in. They work no matter what gene’s in there and they’re designed so that their orientation sends Pol traveling into the insert. When you’re checking cloning products it’s especially important that you get good coverage of the insertion sites because that’s where errors are most likely to occur.
If you have a short gene you might be able to read it all with just end primers (if it’s really short only one may suffice) – but if it’s longer you might need to add additional primers that start in the gene itself (you’ll definitely have to custom-design those since they’re gene-specific not vector-specific)
I usually send DNA from 3-5 colonies of each construct for sequencing – I add the colony to liquid media to let it grow lots overnight, then do a mini prep (alkaline lysis) to purify out the plasmid DNA. Then I use the NanoDrop to figure out its concentration based on its UV absorbance (light-stealing)(the more DNA the higher the absorbance & you can use Beer’s Law to convert between the 2). I want to know the concentration because sequencing companies want a certain quantity of DNA so I need to know how much to add
I calculate so I’m in the recommended range, add one of the primers, & add water to the desired final volume. I do this one per primer. And then I wrap it in bubble wrap, stick it in a 50mL Falcon tube, and send it off! A couple days later I get sequencing results as a chromatograph with peaks representing the different bases, and I check this against what I expect.
antibiotic selection – did I submit a form?
blue-white screening – did I put in *a* password?
colony pcr or analytical digest – is it the right *length*?
sequencing – is it the right *sequence*?
more on recombinant expression: http://bit.ly/2G5N5tY
more on minipreps: http://bit.ly/2MNGJ50