Gateway cloning: a BP reaction, then an LR reaction

• You will end with: a 1,412 bp circular entry clone (BP), then a 1,551 bp circular expression clone (LR) with the gene between attB1 and attB2.
• Time: about 25 minutes.
• Files: GW-Insert.gb, GW-pDONR.gb, GW-pDEST.gb, GW-bad-pDEST.gb (exercise 3 only).
• Before this: nothing; Before you start if the Bench or the sample files are new to you.

Before you click anything, close every tab you do not need. The att sites in these files are invented: see How it works.

Exercise 1 of 3: the BP reaction, an insert into a donor vector

1. Choose File ▸ Open and select GW-Insert.gb, GW-pDONR.gb and GW-pDEST.gb (or drag them onto the window). Each opens in its own tab.

   If instead you see other tabs, close them: the panel takes its molecules from the open tabs.

2. Click the GW-Insert tab.
3. Open its Features tab.

   You see attB1 and attB2 in the list, one on either side of synthGene.

4. Click the GW-pDONR tab and open its Features tab.

   You see attP1 and attP2 in its Features list, on either side of ccdB.

5. Click the Bench tab.
6. Click Gateway at the top of the middle column.

   You see the heading Gateway, with att sites, no enzyme beside it. The panel opens on LR.

7. Click BP.

   You see Choose the attB substrate and the donor vector. A BP needs two attB sites on the first and two attP sites on the second.

8. Open the attB substrate list.

   You see each tab listed with the sites it annotates: GW-Insert — attB1, attB2, GW-pDONR — attP1, attP2 and GW-pDEST — attR1, attR2.

9. Choose GW-Insert as the attB substrate.
10. Choose GW-pDONR as the donor vector.

    You see Entry clone: appear, with Product: 1,412 bp, circular · synthGene, KanR, ori, attL1, attL2, and the only warning: The ccdB cassette of GW-pDONR leaves on the byproduct, so an entry clone that grows in an ordinary strain is the one you want; a ccdB-resistant strain (DB3.1) grows both.

    Why

    The cassette is not in the clone, so a strain that ccdB kills grows the clone and not the donor you started with.

    You see Open clone and no Open byproduct: a linear substrate has no circle to leave behind.

    Why

    The clone is smaller than the donor with an insert added: 1,390 + 422 = 1,812, and the clone is 1,412, because the cassette and the flanks left. Nothing was cut and nothing was joined: the two sites on each molecule swapped the DNA between them.

11. Click Open clone.

    You see a new tab, GW-Insert × GW-pDONR BP, with the Features tab in front. Find synthGene, KanR, ori, attL1 and attL2, each once, and no ccdB.

    • The new tab is 1,412 bp, circular.

    If not, check that attB substrate is GW-Insert and donor vector is GW-pDONR.

    Why

    attL1 starts at base 1,386 and runs 13 bases past the end of the sequence, onto base 1, so the Features tab lists attL2 before attL1 (and the pickers say attL2, attL1). The recombination did not know where the circle starts; the document is the same circle however it is rotated.

    The clone is a scratch document: File ▸ Save if you want to keep it. It remembers how it was made, which the History tab's Made from shows. Leave it open for the next exercise.

Exercise 2 of 3: the LR reaction, an entry clone into a destination vector

1. Click the Bench tab.
2. Click Gateway, then click LR.
3. Open the entry clone list.

   You see the four open tabs with their sites, including GW-Insert × GW-pDONR BP — attL2, attL1, and GW-pDEST — attR1, attR2.

4. Choose GW-Insert × GW-pDONR BP as the entry clone.
5. Choose GW-pDEST as the destination vector.

   You see Expression clone: appear, with Product: 1,551 bp, circular · synthGene, synthetic promoter, synthetic terminator, AmpR, ori, attB1, +1 more.

   You also see the warning The ccdB cassette of GW-pDEST leaves on the byproduct, so an expression clone that grows in an ordinary strain is the one you want; a ccdB-resistant strain (DB3.1) grows both. and two buttons, Open clone and Open byproduct.

   Why

   The panel matched attL1 with attR1 and attL2 with attR2 by number, found the 15 bp core of each pair, and swapped the stretch between the sites. The entry clone is a circle, so both circles of the exchange exist.

6. Click Open byproduct.

   You see a new tab, GW-Insert × GW-pDONR BP × GW-pDEST LR byproduct, 1,698 bp, with ccdB, CmR, KanR, ori, attP1 and attP2.

7. Click the Bench tab.
8. Click Open clone.

   You see a new tab, GW-Insert × GW-pDONR BP × GW-pDEST LR, 1,551 bp, with the Features tab in front.

   • The clone is 1,551 bp and the byproduct 1,698 bp, both circular.

   If not, check that entry clone is GW-Insert × GW-pDONR BP and destination vector is GW-pDEST.

9. In the clone's Features tab, find attB1, attB2, the promoter, the terminator and AmpR.

   You see attB1 at 25 bp (bases 1,540 to 1,551 and, past the origin, 1 to 13), and synthGene at 20 to 379, 360 bp, ATG to a stop, in one piece.

   Why

   LR makes attB, the same kind of site the PCR product started with, and the gene went through two reactions without a base changed.

   The clone and the byproduct are scratch documents; the clone is the one to keep.

Exercise 3 of 3: what a bad design looks like

Two ways for a reaction to fail before it starts. GW-bad-pDEST.gb is GW-pDEST with the second site named attR3, a core that nothing in the insert shares.

1. Choose File ▸ Open and select GW-bad-pDEST.gb.
2. Click the Bench tab.
3. Click Gateway, then click LR.
4. Choose GW-Insert × GW-pDONR BP as the entry clone.
5. Choose GW-bad-pDEST as the destination vector.

   You see, in red, The sites do not pair: GW-Insert × GW-pDONR BP has attL1 and attL2, GW-bad-pDEST has attR1 and attR3. Each number recombines only with its own. There is no product line and no Open clone.

   Why

   attL2 has no attR2 to meet. The panel does not guess that attR3 is close enough; in the lab it would simply not recombine. This is what you have if you pick the wrong destination from a catalogue.

6. Choose GW-Insert as the entry clone instead, and GW-pDEST as the destination vector.

   You see GW-Insert has no attL site annotated. A LR needs two, flanking the DNA to move; annotate them, or open a file that does.

   Why

   The PCR product has attB sites, and an LR starts from attL. This is the usual mistake of leaving out the BP, or of opening the wrong tab.

7. Click BP.
8. Choose GW-Insert as the attB substrate, and GW-pDEST as the donor vector.

   You see GW-pDEST has no attP site annotated. A BP needs two, flanking the cassette they replace.

   Why

   A destination vector has attR sites, so it is not a donor; the panel says which kind it was looking for.

9. Choose GW-pDONR as the donor vector again.

   You see the BP is back.

Done when:

• The entry clone is 1,412 bp, circular, with attL1 and attL2 and no ccdB.
• The expression clone is 1,551 bp, circular, with AmpR, attB1 and attB2 and no KanR, CmR or ccdB (the byproduct has all three of those and no AmpR).

How it works

The att sites in these files are invented. PlasmidPop carries no att sequences: it reads the sites a file annotates (attB1, attP2, attL1, attR2 and so on) and pairs them by number. Here each site is a 15 base core with a short arm on each side, so the sizes (25, 55 and 40 bp) are not those of the real sites, and none of the sequence is. With a real donor, entry or destination vector, which labels its own sites, the steps are the same. The app does not design primers that add attB sites to a PCR product, so the insert here is supplied as the product, with its attB sites already annotated; in the lab you would put the attB tails on the primers yourself. A plasmid that does not label its sites gets a message, not a guess. All four files are synthetic examples.

The seven words of this page:

• att site: a short stretch where the integrase swaps DNA. There are four kinds, attB, attP, attL and attR, and each carries a number (attB1, attB2).
• Core: the bases two partner sites share. The panel finds the core itself (the longest stretch the two sites have in common) and crosses over in its middle. Here it is 15 bp.
• BP reaction: attB substrate × donor vector (attP) gives an entry clone (attL) and a byproduct (attR).
• LR reaction: entry clone (attL) × destination vector (attR) gives an expression clone (attB) and a byproduct (attP).
• Numbers pair: attB1 recombines only with attP1, attL1 only with attR1. The two numbers on a molecule are what give the insert its direction.
• ccdB cassette: a gene that kills an ordinary E. coli strain. It sits between the two sites of a donor and of a destination vector, so it leaves with the byproduct and the clone you want is the one that grows.
• Selection marker: each vector carries one for its backbone. In a BP the clone keeps the donor's, in an LR the destination's, and the other comes away on the byproduct.

The four files:

GW-Insert is a PCR product, so it is linear. The donor and the destination carry the ccdB stand-in between their sites; the destination's cassette also carries a CmR stand-in, as real destination vectors do. You never type a sequence.

The sums of exercise 1: the insert contributes 397 bp (422 less 12 bp before its attB1 crossover and 13 bp after its attB2 one); the donor contributes its backbone, 1,015 bp (1,390 less the 375 bp cassette). 397 + 1,015 = 1,412. The 400 bp that are not in the clone are the 375 bp cassette and the 25 bp of the substrate's flanks. A linear substrate has no circle to leave behind: its two ends come away as loose pieces, and the panel does not make a document of them. The warning talks about a byproduct the panel did not give you: it says where the cassette went, whether or not the reaction made a document of it.

What the exercise 1 clone holds, beyond what the steps showed:

• attL1 and attL2 are 40 bp each: half of an attB's bases and half of an attP's. They are different sequences (their cores differ), which is what keeps the insert's direction.
• The gene is whole, 360 bp, synthGene at 20–379, between the two sites: attL2 follows it at 386–425, and 6 bp separate attL1 from it.
• KanR (the donor's marker) is on the clone, on the reverse strand as in the donor.

The sums of exercise 2: 1,412 + 1,837 = 3,249, and 1,551 + 1,698 = 3,249. Every base that went into the reaction is in one circle or the other; a recombination exchanges DNA and loses none, unlike a digest and a ligation, and if the two sizes do not add up, something else was open. The clone is the entry clone's 397 bp between its sites plus the destination's 1,154 bp outside its cassette (1,837 less 683); the byproduct is that 683 bp cassette plus the entry clone's 1,015 bp backbone. The destination loses the 683 bp between the middles of its two cores (ccdB, CmR and the spacers) and gains the 397 bp of the entry clone's insert piece, while the entry clone's backbone, with KanR and the origin, goes the other way onto the byproduct.

What the exercise 2 products hold:

• attB1 and attB2 are 25 bp each, the very bases the PCR product had. The byproduct's attP1 and attP2 are 55 bp, the very bases the donor had.
• synthGene is whole (360 bp), with 6 bp between attB1 and its ATG, and 12 bp between attB2 and the terminator.
• The clone's features are synthGene, synthetic promoter, synthetic terminator, AmpR, ori, attB1 and attB2, each once. The byproduct's are ccdB, CmR, KanR, ori, attP1 and attP2. The markers are the swap the selection uses: ampicillin for the clone, and the ccdB and chloramphenicol genes on the circle that does not grow.
• Each new site is half of each parent's: an attB is half of an attL and half of an attR, which is why the numbers and not the names say what pairs. The Product line lists synthGene first, before the promoter, because the document starts where the crossover wrote it; along the circle the order is promoter, attB1, synthGene, attB2, terminator.

PlasmidPop reads the att sites from the annotation and carries no att sequences, and it does not design attB primers or annotate a PCR product's attB tails. To use your own gene, make the PCR product in the lab (or add the attB sites to the sequence yourself) and annotate its two sites attB1 and attB2; see Features. There is no NCBI gene path in this tutorial: a gene from NCBI would need those sites added by hand first, and nothing in these pages contains a third-party record.

Next

Gateway on the Bench in the user guide also covers a vector annotated on the other strand, the check against a ccdB gene on the wrong circle, the frame warning for a tag in the backbone, and why a multisite reaction is done a fragment at a time. The design note is item 48 (docs/design/48-gateway.md). Back to the tutorials index.