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in this video we will see what is
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Gateway cloning and how it is different
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from classic restriction enzyme based
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cloning I will go over some basic
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knowledge and Link it to simple Gateway
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cloning strategy as well as complex
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Gateway methods I will discuss specifics
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of LR and BP reactions and dive into
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their steps in a bit of detail finally
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we will see what makes Gateway great and
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some reasons to stay away from it
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Gateway cloning has been around for more
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than two decades and is commercialized
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by invitrogen this means you can buy it
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as a kit it is a method to clone without
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using restriction enzymes traditionally
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in restriction based cloning you start
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with a vector a plasmid backbone into
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which you would like to insert something
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so you digest and perhaps purify the
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vector in parallel you prepare the
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insert which could be a PCR product or
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perhaps a piece of DNA from another
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Vector which means you are subcloning
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depending on your strategy you digest
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maybe you blunt and purify the insert
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and combine it with the backbone this
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liation gets transformed selected
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screened and eventually validated
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possibly for orientation if you do blunt
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cloning I have separate video that
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discusses these things in much more
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detail these steps from start to the
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liation are labor intensive Gateway
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cloning replaces all these upstream
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manipulations and liation steps in one
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super quick reaction so what is
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happening in this Gateway cloning relies
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on at site recombination at sites are
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attachment sites and they are DNA
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sequences here's some context to
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attachment sites think of a happy
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bacteria that is swimming but out of
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nowhere a bacteria phage Lambda attacks
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the bacteria the infection leads to
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viral DNA injection into the bacteria if
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you know a little bit about the virus
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life cycle the viral DNA can integrate
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into the bacterial genome this
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integration occurs due to recombination
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of the attachment sites between the two
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genomes so both virus and bacteria DNA
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carry at sites Fage at sites are called
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at P bacterial at sites are called at B
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depending on how you define them they
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range from 25 to 300 nucleotides in
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length a typical atsite is split into
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two portion say p and p Prime and the
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middle recombination spot is called o
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likewise you have B and B Prime and the
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middle spot is called o the O is the
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recombination spot between at p and at B
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let's actually understand how this viral
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integration works and how at site
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recombination is involved in this the
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virus DNA as we said has the ATP site
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I'm drawing the virus DNA as circular
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for ease of understanding instead of
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calling these two halves of ATP as p and
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p Prime let's name them one and two the
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ATP of pectoral DNA likewise has has B
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and B Prime but let's call them three
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and four when the viral DNA needs to
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integrate into the host genome it makes
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an enzyme called integrace the Integra
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is recognize at B and at p and cause a
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recombination to occur between them this
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recombination between the two ad sites
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is called a BP reaction recombination
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event occurs at the O region between the
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two at sites if you start from the
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perspective of site three the
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recombination brings number number two
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in front of three and now this entire
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viral DNA until number one comes along
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with number two but now the number one
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gets attached to number four from number
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four we get back to the bacterial DNA
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which comes full circle to number three
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from this BP reaction we see that the
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virus DNA has integrated into the
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bacterial DNA and that is the proage
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life cycle of the virus also just to get
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back to these chimeric BP sites this BP
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site is called at L and this PB site is
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called at R these stand for left and
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right you may come across some old
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literature that calls them Bop or po but
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I will use at L and at R terminology now
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that we understand this let's look at a
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simple Gateway cloning this uses 2 ATP
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and 2 at B sites assume that our final
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Vector will be this blue Vector carrying
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a conomy marker it carries two at P
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sites with a ccdb suicide gene in the
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middle the second Vector carries two at
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B sites and it has the DNA of Interest
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we want to clone in the middle this is a
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donor Vector just like this DNA of
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Interest being in the plasmid form you
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can also have the DNA of interest from a
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PCR but in that case you will have to
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add add B sites to the primer for
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Simplicity I will restrict the
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discussion to the plasmid form of the
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donor Vector for Gateway cloning you
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take these two plas and combine them
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with integrases in a BP reaction this
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causes recombination between the ATP and
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at B sites so now we can redraw the
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final vector and let's start with this
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part of at P which gets switched with at
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B and then the DNA I continues until you
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have half of at B which gets recombined
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with ATP and this comes back full circle
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essentially we have swapped DNA into the
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final Vector like this you can also we
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draw the donor Vector which instead of
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DNA I now contains the ccdb Gene and
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like we saw earlier you can annotate
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these new chimeric sites as at R and at
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L note that all of this happens in vitro
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and following this reaction it is ready
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to be transformed and the bacteria is
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grown in kyin because the final Vector
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containing our DNA I has kyin which
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means any bacteria carrying this other
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plasmid will die so in in this simple
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Gateway cloning we have learned about
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one simple rule at p and at B can
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recombine but at p and at P do not
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recombine likewise at B and another at B
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cannot recombine if these two were true
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then bacterial and viral DNA during
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infection process will recombine by
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themselves and that will mess up
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everything all right so we have switched
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this internal portion between the two
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plasmids and this is a recombination
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mediated exchange but we saw that during
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infection the viral DNA integrates into
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the host genome but in Gateway cloning
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we talk about exchange so where is the
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integration event in this Gateway
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process let's break this process down
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and understand stepwise how integration
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can lead to exchange we have two at p
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and two at BS for Simplicity let's label
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the halves as 1 2 3 4 5 and 6 and 7even
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and 8 and to to make this simple imagine
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that this pair of at gets recombined
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first Also let's assume that this second
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pair is not used until the first
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reaction is complete and we will focus
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on the final vector and anchor at this
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site one as we saw before anchor site 1
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recombines with site six site 6
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continues into site 7 and site 8 if we
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continue we see this entire plasmid
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comes back to site five site five gets
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recombined with site two and this site
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two continues to site three and four and
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all this circle backs to chomy this is
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an intermediate plasmid where only one
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BP reaction has occurred and we have
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integrated the donor Vector into the
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final Vector in this we have one at r
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one at L one at B and one at P the at B
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and P can further undergo a second BP
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reaction to understand this next step it
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is best to redraw the plasmid in a
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different configuration this is done
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such that the B and P are next to each
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other you can pause this video and try
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to draw this yourself as well in the
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second BP reaction the site 7 gets
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recombined with site 4 which comes back
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full circle to conomy this releases our
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dnf interest in the conomy plasmid the
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second recombination occurs from site
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three to site 8 which Circle backs to
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illin with the chimeric site 5 and 2 and
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the ccdb gene you can label all these
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sites as at R and at L if you look
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closely these are exactly the same
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product we had originally produced so
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this way sequential BP reactions can
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integrate and exchange the DNA with ccdb
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in the donor and the final Vector okay
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now we can move to the second part of
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the simple Gateway cloning you remember
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this proage structure we discussed a few
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minutes ago where you get the full at L
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and at R another aspect of the virus
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life cycle is that propage can move to a
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litic cycle where it is possible that
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the viral genome is excised out of the
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bacterial genome how does that happen to
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understand that we have to redraw the
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configuration of this genome in the
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following way during litic phase some
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exision integrases and other enzymes are
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expressed these enzymes can recombine at
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R and at L looking at site three it will
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recombine with four and this gives you
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the bacterial DNA likewise number one
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recombines with number two which
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releases the viral DNA previously we
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have noted BP reaction that occurs
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between at B and at P this excision
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occurs between at L and at R so we call
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this an LR reaction from the LR reaction
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we can recover the at p and at B sites
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just like B and P we can have another
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variant in our simple Gateway cloning
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where we use two at R and two at l s
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sites in this style of Gateway the final
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Vector has at l s sites for example and
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likewise the donor Vector will have
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compatible at R sites and these two
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vectors will use another set of specific
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enzymes and this will be an LR reaction
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and the recombination occurs in a
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similar fashion except that the final
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forms have ADD B and add p in the
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products this is yet another example of
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recom combination mediated exchange the
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rules for LR reactions are that L and R
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can recombine but two L's cannot and
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likewise two at RS cannot the
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interesting bit to note is that the at B
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containing DNA I in the final Vector can
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become a new donor for a new BP reaction
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if you wanted to use it now that you
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understand the simple Gateway cloning
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let me give you a snapshot of some
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complex ways to do Gateway cloning this
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is called a multi-site Gateway simple
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Gateway has at B and at P compatibility
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however there are variants of at B and
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PS for instance you have at B1 and at P1
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which are compatible with each other but
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B and P1 are not compatible and likewise
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B1 and P are not compatible just like B1
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and P1 you also have other variants on B
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and PS suffice to say that their
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products are likewise also variants and
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one variant pair is not compatible with
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another variant pair let's do this with
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an example where you're trying to clone
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a promoter and an ORF into a plasmid at
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the same time let's assume that the
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final Vector has at R1 and at R2 then
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you have the two donors one containing
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the promoter between at L1 and at R3 and
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the second donor containing the ORF
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between L2 and L3 in the multi-site
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Gateway you mix all these three vectors
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in one reaction and cross Vector
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recombination can occur in the presence
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of specific LR reaction enzymes each
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variant gets recombined independently if
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you solve this puzzle step by step you
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will note that the promoter flanked by
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B1 and B3 the ORF is flanked by B3 and
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B2 and this entire configuration is
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present in the final canamy inor if you
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want step-by-step details on this
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reaction it is available on my patreon
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along with the reference material used
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to make this video this multi-site
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Gateway doesn't just stop here you can
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come in with a new Vector containing P1
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P2 and P3 and switch out the ORF if you
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want it to or even the promoter using a
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donor Vector containing P1 and P3 this
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brings us to the last segment of this
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video the advantage of Gateway is that
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it saves time but this comes at a very
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high cost but it is quite efficient it
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reaches very high throughput and it is
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very flexible as we saw from the
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multi-site Gateway the process is
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virtually scarless since no restriction
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enzymes are involved multi-site Gateway
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is also very powerful because it saves
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you sequential cloning steps if you were
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to use classic enzyme based cloning
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methods the disadvantage is that
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restriction enzymes are not involved so
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you're actually stuck in this Gateway
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cloning ecosystem and the cost of
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switching out is very high we did say
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that the lack of restriction enzymes
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leaves no scar but you still have to
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clone your DNA into a donor Vector with
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at sites so it is not 100% enzyme free
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this becomes very complicated if you
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were starting from a scratch and you
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have to build multiple donor vectors
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containing these complex sites so
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restriction enzymes are still required
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in the beginning stages of the Gateway
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cloning there's one concern with Gateway
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expression vectors like the one here
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containing a promoter and an ORF there
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is this undesirable ad B site you find
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in the middle of the ORF and promoter
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and it may be that it interferes with
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the expression Dynamics these are some
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points I would like you to consider when
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it comes to Gateway cloning so ATP and B
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are involved in Gateway a similar
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ecosystem of cloning is called uni
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vector which works like Gateway but it
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relies on locks spe sites so both of
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these are recombination mediated
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cassette exchange cloning methods there
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is an orthogonal approach roach that
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uses homologous recombination some
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methods in this space are infusion magic
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sefc perhaps you will see them in a
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separate video and maybe I will see you
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there as well
