Feeling blue? These bacteria are too – and that makes us molecular cloners feel blue because it means our gene didn’t get into the circular piece of DNA called a plasmid which we put in there, so the bacteria can’t make copies of the gene for us. But the good news is, the blue-white screening system makes it easy to tell – so we can avoid the blue and instead choose a white colony, where molecular cloning went well!
An overview in rhyme, then it’s details time! If the plasmid vector your insert hacks, blue-color-making protein the bacteria lacks! The plasmid has the sequence the bacteria needs, but you wouldn’t know this if your cloning succeeds. When you insert the gene, that sequence you break & the functional protein the bacteria never make. If the colony’s white you know all went right, but if the colony’s blue, you need to redo
Each of the dots on this plate is a clump of genetically-identical bacteria, which we call a colony. They’re genetically identical because bacteria reproduce by doubling their insides (including their DNA) and then splitting in half, giving a full set of everything to each daughter cell.
In addition to their own DNA, these bacteria have an “extra” circular piece of DNA called a plasmid. I know that these bacteria have the plasmid I want because the plasmid has antibiotic resistance genes that allow it to survive even though I spiked the bacterial food (media) with the corresponding antibiotics. This is a form of SELECTION – only bacteria with the plasmid inside can grow on these plates.
But, apart from allowing for selection against random bacteria trying to grow on our plates, the plasmid is really just there to act as a vehicle, or VECTOR, to allow us to insert “any” gene we want into the bacteria – so, for example, they can make lots of copies of it for us. Since we’re recombining pieces of DNA when we do the vector/insert combo a form of RECOMBINANT DNA. And since the bacteria make lots of copies of it, and when they divide each daughter cell is a genetic clone of the parent cell, we call this general process MOLECULAR CLONING.
The basic gist of molecular cloning is: stick a gene you want to study into a circular piece of DNA called a plasmid vector & stick that plasmid into bacteria to make more of that gene &/or the protein it codes for (possible because the genetic code is universal so any organism can read it). It’s a powerful tool that’s revolutionized molecular biology & biochemistry. BUT it doesn’t always work right, so we need to check that
- the bacteria actually took in the plasmid AND
- our gene actually got inserted into the plasmid
We can use SELECTION MARKERS like antibiotic resistance genes to check for the presence of the plasmid (1) e.g. if plasmid has Ampicilin (Amp) resistance gene but host bacteria don’t, only bacteria that have the plasmid can grow in presence of Amp,sSo any colonies of bacteria that grow have the plasmid. BUT does that plasmid have your gene in it (2)?You still don’t know, but there are a few ways to find out.
A couple options we’ve looked at are the DIAGNOSTIC DIGEST (aka analytical digest) http://bit.ly/30Npa8o & COLONY PCR http://bit.ly/39cADmK . These either CUT (in the digest) or COPY pieces of (in the PCR) the plasmid differently if your gene is present or absent and this gives you different size DNA pieces. In order to see these pieces you have to separate them by size using agarose gel electrophoresis. And for the digest, you have to purify the plasmid DNA before you can even test it. So, while these methods work (for the most part), they can take a while.
BLUE-WHITE SCREENING (B-W screening) is a way to check for presence of an insert without even having to touch the colonies! You just have to look!
It uses protein called BETA-GALACTOSIDASE (B-gal), which is an enzyme that catalyzes (speeds-up) the hydrolysis (water-based breaking up) of LACTOSE (a disaccharide (2 linked sugar units)) into the monosaccharides (individual sugar units) GLUCOSE & GALACTOSE. The GENE for B-gal is called lacZ.
B-gal functions as a homotetramer (4 identical copies stuck together). First it forms dimers (pairs) & then those pairs pair to form tetramers. You need all four because parts of the different copies “cross over” to contribute to the “active sites” where the functioning occurs
This pair-pairing (dimer to tetramer) occurs using the beginning part of the protein (N-terminus). if you remove this part, dimers can still form, BUT tetramers can’t, so functional protein can’t be made. BUT you can restore this beginning, by adding the needed part, the alpha (a) peptide, back “in TRANS” (as a separate molecule). This is known as a-complementation.
In B-W SCREENING you use bacterial host cells have a shortened B-gal (LacZΔm15) that’s missing some of the protein’s first 41 amino acid “letters” (11—41). (Δ is the Greek letter “delta” and, in protein/gene jargon, it’s often used to indicate missing parts). These LacZΔm15 are missing the N-terminus part & only have the “leftover” ω-fragment that can’t form tetramers.
To make it form tetramers, it needs those first letters – the “α-peptide” – & the plasmid has that to offer in trans (plasmid’s way of paying rent?). So you need both in order to make B-gal. But how do you know if B-gal is actually being made?
B-gal’s usual products (glucose & galactose) aren’t colored, so we can’t see them. BUT B-gal mostly cares about the galactose part & isn’t too picky about what’s on the other side of the glycosidic bond. So we can swap the glucose for something that will give us a product we can see.
X-gal (5-bromo-4-chloro-3-indolyl-b-D-galactoside) has galactose connected to 5-bromo-4-chloro-3-hydroxyindole. β-gal splits it up into galactose & 5-bromo-4-chloro-3-hydroxyindole which dimerizes (pairs up) & oxidizes to 5,5′-dibromo-4,4′-dichloro-indigo which is insoluble & BLUE! So the colony turns blue.
That doesn’t seem too useful though… We only want to grow colonies with our plasmid in the first place, which is why our plasmid ALSO has a selection marker in the form of an antibiotic resistance gene. So just having plasmid-containing colonies turn blue would be redundant – it’d tell us what we already know – the plasmid’s there.
The real power of blue-white screening is making them NOT BLUE. To do this you use plasmids designed to have a multiple cloning site (MCS) (the place you put in your gene) INSIDE the gene for the α-peptide. This way, inserting your gene interrupts the α-peptides’s gene, so the bacteria can’t make functional α-peptide -> can no longer complement the ω-fragment -> can’t make functional B-gal -> can’t make blue product from X-gal -> colony remains white
- WHITE COLONIES have insert (in right place)
- BLUE COLONIES don’t have insert or have it in the wrong place
⚠️whiteness indicates that SOMETHING got inserted. BUT you don’t know that that something’s the something you want -you’d have to sequence it to be sure
Of course, this B-W screening only works if β-gal is made! & bacteria don’t want to waste time & resources making β-gal if they can’t use it – so we have to trick them into using it
When lactose isn’t around, a REPRESSOR protein sits on & “hides” the part of the β-gall gene (lacZ) where the DNA-to-RNA copier RNA POLYMERASE (RNA Pol) binds to start making an mRNA copy of the gene that can then get read by ribosomes and translated into the protein. So, NO lactose -> no β-gal mRNA made -> no β-gal made (not even the monomers…)
BUT when lactose is present, B-gal “moonlights” as a transglycosylator – it converts lactose into ALLOLACTOSE by changing the glycosidic linkage site between the glucose & galactose units (just shifts things around a little to give you an isomer of lactose – same atoms, different linking). Allolactose binds the repressor causing the repressor to change shape (undergo a conformational change) & fall off -> RNA Pol binds -> β-gal mRNA made -> β-gal made
So you’d think, why not just add some lactose, or allolactose if we want to do B-W screening? Problem is, β-gal can also hydrolyze (split) our de-repressor allolactose. So, we’d have to constantly add more if we wanted to have continuous β-gal making. So, instead, we trick the bacteria by using IPTG Isopropyl (β-D-1-thiogalactopyranoside). This is an allolactose analog (mimic) that can also de-repress the lac promoter, so β-gal is made. BUT unlike lactose it doesn’t get hydrolyzed b yβ-gal, so it sticks around.
note: if you move the lac promoter in front of a gene for something else, you can use IPTG to trick the bacteria into making that something else on cue. we looked at such “induction” in yesterday’s post on recombinant protein expression in bacteria: http://bit.ly/3cPVhLO
So, in practice, it works something like this.
- put your gene in the plasmid. (molecular cloning step)
- put the plasmid in the bacteria (transformation step)
- plate the bacteria on an agar plate (you’re classic “Petri dish” w/food, antibiotic, IPTG, & X-gal
- stick it in a nice warm incubator
- wait for the bacteria to grow
- wait for blue color to show
If you come back the next day & all your colonies are white, don’t get too excited yet – It takes a while (16-20h) for β-gal to be expressed (remember it has to get de-repressed & everything first) & get to work. So the blue starts showing up gradually. Sometimes it’s hard to tell early on, but the more you work w/this system, the more of a “sense” you get for it. The white ones usually look kinda “different” – they grow bigger & look “goopier”
If you come back the next next day & all the colonies are still white, definitely don’t get excited – get suspicious, because cloning is rarely that efficient. Did you forget the IPTG? the X-gal?
note: X-gal is the “classic” chromogenic (color-producing) B-gal substrate, but you can change up that indoxyl part to change the absorbance and therefore the color you see. Our lab uses Bluo-gal, which is really similar but doesn’t have the chlorine . This gives a darker blue product that’s easier to see. You can also do things like move the Cl to a different position to get Magenta-gal (5-bromo-6-chloro-3-indolyl-β-D-galactopyranoside). Remove the Br from that and you get the more salmon-colored (thus aptly named) Salmon-gal (S-gal, 6-chloro-3-indolyl-β-D-galactopyranoside). The color differences come from the different molecular arrangements absorbing different wavelengths of light. And if you want to know more: http://bit.ly/2RDLVY4
Technical terminology talk time: Color chemistry-ly speaking, “DYE” is usually used for *soluble* colored things whereas “PIGMENT” implies *nonsoluble*. Since X-gal’s product is insoluble, it’d technically be a *pigment* not a *dye* but in biology, we tend to use the terms interchangeably
Technical terminology talk time 2: B-W is a form of SCREENING as opposed to SELECTION. SELECTION (like w/antibiotic resistance gene) does all the work for you (all the products are “good” in the aspect you selected for) BUT w/a screen you have to do some work (even if it’s just looking) – the “duds” are still there, they’re just easier to see.
It’s like you’re looking to hire some people – with screening, you go through all the apps & separate them into yes/no piles but keep both for someone else to deal with. With selection, you toss the no pile, so the next person only sees the yeses.
Now for a couple caveats: Firstly, screening isn’t always yes/no. You can have “maybes” like if you’re screening for drugs that might work for something & some work really well, some don’t work at all, but some are “potentials”
Secondly, only certain bacterial hosts & plasmids are designed for blue-white screening. Some that are:
- hosts: XL1-Blue, DH5α, DH10B, JM109, STBL4, JM110, & Top10
- plasmids: pGEM-T, pUC18 and pUC19, & pBluescript
more on topics mentioned (& others) #365DaysOfScience All (with topics listed) 👉 http://bit.ly/2OllAB0