Wednesday, December 2, 2009

An alternative cloning strategy: yeast recombinational cloning
As part of my Ph.D thesis, I have to generate a lot of transcriptional fusions (constructs in which a promoter of choice is cloned in front of a reporter gene in order to evaluate transcriptional regulation. Such plasmids can then be transformed into your model organism to study this regulation in vivo).

Traditionally, this involves amplifying the region of interest (in my case a promoter region) by PCR using primers that harbor the recognition site of particular restriction enzymes. Sometimes you can use the PCR product directly in a digestion reaction with the proper enzymes (although it’s recommended that you column purify your PCR product first) in order to clone it into the vector of interest, but many prefer to clone it first into pGEMT. In order to do the latter, you’ll have to do a TA tailing, as high fidelity enzymes (used for cloning) typically produce blunt ends. Once again, it’s recommended that you column purify your PCR product before tailing.

Once tailed, you can clone the PCR product into pGEMT and transform E. coli in order to propagate the plasmid, which can now be sent out for sequencing. After everything checks out, you would have to set up a digestion reaction in order to get the segment out of pGEMT and clone it into the final plasmid. After that digestion, and in order to get this segment into the final plasmid, you would digest the target plasmid with the same enzymes, set up a ligation reaction and then use an aliquot of the reaction to transform E. coli.

Ideally you’ll use two different enzymes for target plasmid linearization, which would avoid plasmid re-circularization, but sometimes things are not that easy. If you can only use one enzyme for linearization (due to plasmid design, enzyme availability in your lab, etc.) then you are going to have to dephosphorylate the linearized plasmid (typically using Calf intestinal phosphatase) after digestion to avoid re-circularization. Some phosphatases can be heat inactivated, but others can’t. In any case, after digesting and dephosphorylating your plasmid, you’ll have to do a phenol:chlorophorm extraction followed by ethanol precipitation to get your dephosphorylated plasmid, which can now be used for ligation with the segment of interest. After setting up this ligation, an aliquot is used for transformation into E. coli.
You can then check your plasmid by colony PCR of the antibiotic-resistant colonies resulting from the transformation.

At this point you’ll realize that generating several constructs following this protocol can take some time. This strategy, then, does not fit my cloning needs.

We’ve taken a different approach to satisfy most of our cloning requirements, which makes use of my favorite organism: Saccharomyces cerevisiae.

The cloning strategy we use routinely in our lab is called Yeast Recombinational Cloning (YRC), a strategy which has been around for some time (Ma et al., 1987) and has been refined over the last ~13 years (Oldenburg et al., 1997; Gibson, 2009).

The concept behind this strategy is simple: if the segment you’d like to clone into a particular plasmid bears homology to defined plasmid sequences, you can directly “ligate” it into the linearized vector by in vivo recombination: yeast machinery will take care of it. This alleviates the need for an in vitro ligation reaction.

Initially, it was shown that a DNA restriction fragment containing appropriate sequence homology could serve as a substrate for such “recombinational repair” (it’s called repair, as you’ll repair the gap in the linearized plasmid). However, an important advance in the use of such recombination-based methods for gene cloning in yeast, involved the use of PCR (rather than restriction assays) to generate the DNA fragment to be used.

As it was shown that the length of sequence homology needed to promote efficient recombination between the segment of interest and the plasmid was small (~20-40 bp), it was quickly realized that these sequences could be included as part of PCR primers used to amplify a segment of interest. This “recombination-mediated PCR targeting” is very efficient and I consider it now one of my favorite techniques.

So, how do you generate your plasmid through YRC? The linearized target plasmid containing a selectable marker (e.g. URA3 ) is co-transformed into yeast with the PCR fragment of interest: this fragment has 20-40 nt of homology at each end to the region of the plasmid at which recombination is to occur. These nucleotides were added to the fragment as part of the primers. You don’t even have to purify the PCR product before transformation; an aliquot taken directly from the PCR reaction tube works fine. By homologous recombination, some of the cut plasmids are recircularized (due to the integration of the segment of interest into the plasmid) and the plasmid can now be propagated in yeast. Recombinants are then selected as Ura+ transformants (See figure 1).

FIGURE 1. Basic outline of yeast recombinational cloning. Figure based on the one at the “Yeast Model Systems Genomics Group” website.

You can then do yeast colony PCR on the Ura+ transformants to check for your plasmid. Note that the linearized plasmid will not lead to Ura+ transformants, as such a plasmid cannot be propagated.

This protocol may take longer (in days) than the traditional approach outlined at the beginning of this post (as you'll have to wait for the transformed yeast to grow in selective media), but it has significantly less steps, uses less restriction enzymes (as you'll only use them to linearize the target plasmid), uses no ligase, does not require any sort of purification and the only methods involved are digestion, PCR and yeast transformation (the latter is very simple, efficient and requires reagents typically found in a molbio lab. We use the Liac/SS Carrier DNA/PEG method with excellent results).

Further, this approach is more amenable to high-throughput cloning than the traditional strategy (See Colot et al., 2006).

In addition, homologous recombination in yeast can be used to build complex constructs from multiple overlapping constituent parts (see figure 2). Generating such constructs through traditional approaches would take considerably longer. Note that the target vector doesn’t necessarily have to be the final vector. You can use YRC to assembly this complex construct and then the DNA insert can be cut out of the yeast vector and placed into any other vector.

FIGURE 2. Multiple overlapping segments can be cloned into a target plasmid (in this case a shuttle plasmid) through homologous recombination in yeast.

OK, enough about the methodology. How is this strategy used for the generation of my transcriptional fusion constructs? The target plasmid we use is linearized just upstream of the coding region of a reporter gene. Due to proper primer design, the PCR amplicon of the promoter of interest bears homology to particular plasmid sequences that allow its integration upstream of this reporter gene through homologous recombination. The particular target plasmid that we use can be propagated both in yeast and bacteria (i.e it’s a shuttle plasmid) and can also be transformed into our model organism, where by homologous recombination, will integrate in a particular locus in the genome so that we can study the promoter’s biology in vivo.

So, in summary, we have generated a yeast strain which contains a plasmid with our promoter of interest controlling the reporter gene. Now, it’s time to obtain that plasmid in working concentrations, sequence it and then transform it into our model organism.

How do we do this? How do we get the resulting plasmid out of yeast? That will be the matter of a follow-up post, so stay tuned!

Some of the articles discussed in this post:

MA, H., KUNES, S., SCHATZ, P., & BOTSTEIN, D. (1987). Plasmid construction by homologous recombination in yeast Gene, 58 (2-3), 201-216 DOI: 10.1016/0378-1119(87)90376-3

Oldenburg KR, Vo KT, Michaelis S, & Paddon C (1997). Recombination-mediated PCR-directed plasmid construction in vivo in yeast. Nucleic acids research, 25 (2), 451-2 PMID: 9016579

Gibson, D. (2009). Synthesis of DNA fragments in yeast by one-step assembly of overlapping oligonucleotides Nucleic Acids Research, 37 (20), 6984-6990 DOI: 10.1093/nar/gkp687

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Francisco Barriga said...

What you described initially is quite accurate regarding the steps involved (although most people use gel band purification kits instead of phenol/chloroform extraction) I just have two things to point out:

1. If you´re lab uses yeast then YRC is the most straightforward strategy to clone. In my particular case, yeast is not an option and we have to stick to "old school" cloning and to recombination based cloning in bacteria.
The principles are the same regarding the overlap of sequences, the only thing is that you co transform your linearized DNA and plasmid into recombination competent bacteria (as in YRC). Evenmore, there are at least 2 commercial options ofr recombination based cloning: the Gateway system from Invitrogen and the In-Fusion system by BD. Once again, for non-yeast users I recommend to take a look at them.

2. Recombination saves you a lot of steps, that´s for sure, yet In my hands I have found that some regions you just cannot amplify with primers that have a 30-50 bp overhang, so this is just to say to be careful with the primer design (as you always should) and hopefully the locus you need is not very long so the PCR is more efficient.

That being said, recombination based cloning is, in my view at least, a very nice tool that can replace the tedious steps Alejandro mentioned. My impression is that when synthetic biology reaches reasonable prices, you´ll just synthesize the nucleotides of interest and probably our children (may they be molecular biologists..) will never have to go through this again

Alejandro Montenegro-Montero said...

Hi! Thanks you for reading such a lengthy post :-)

Regarding your comments...

I didn't include the gel band extraction/purification step in the last part because it serves the same purpose for what I was trying to say: there is an extra purification step. You are certainly right, anyway. Lots of people do that nowadays.

In my particular case, yeast is not an option

Yeast is always an option.... the target plasmid need not be the final plasmid

Regarding Gateway technolgy, it is expensive, and thus, prohibitive for a budget-limited lab. A typical topo reaction is around ~14 euro and the LR-reaction, 5 euro. Imagine using such technology for generating hundreds of clones.

In addition, and in my opinion, YRC is better than Invitrogen's Multisite technology for making complex constructs (see figure 2). It's way more flexible.

Now, regarding recombinational cloning in bacteria, that's a fascinating area, although we haven't looked into it yet at our lab.

For those interested, see:

J. Proteome Research. 3:582-586 (2004)

Nucleic Acids Research. 21:5192-5197 (1993)

Nucleic Acids Research. 21:3601-3602 (1993).

In my hands I have found that some regions you just cannot amplify with primers that have a 30-50 bp overhang, so this is just to say to be careful with the primer design (as you always should) and hopefully the locus you need is not very long so the PCR is more efficient

Luckily this has never been an issue in our lab. Around 90% of the designed primers worked on the first try without any PCR tweaking using normal Taq, and the remaining 10% worked with a high fidelity polymerase. My products are usually in the range of 2500-3000 bp.

Finally, regarding synthetic biology, yeast-based cloning can be the way to go. See Gibson, 2009.


Nikolai said...

Though I haven't tried YRC myself, I'm not sure that traditional cloning is as difficult as you make it seem. Let's look at how long cloning takes:

1) Amplification of region of interest: 1.5-2 hrs if using a highly processive enzyme like Phusion

2) Cleanup of PCR reaction: 0.5-1 hr w/silica column-based kits (why are you still doing phenol/chlorophorm/ethanol cleanups?)

3) Double-digest of both the PCR product and plasmid: 0.5-2 hrs

4) De-phosphorylation of the 5' ends of plasmids: 0.5 hrs

5) Gel-purification of both plasmid and insert: 1-3 hrs (the gel running time can be drastically cut by using Invitrogen's E-Gels)

6) Ligation + drop dialysis + electroporation + outgrowth in non-selective medium: 3 hrs

7) Growth on selective media: overnight.

Unless I'm missing a step this should take 7-11.5 hrs of real work. So realistically you might spend one day preping the plasmid and insert and an afternoon ligating and plating, so that the whole thing takes three days, including the overnight. All of the steps prior to plating are easily scalable to a 96 well format, so you should be able clone a small library of promoters in less than a week. I've never worked with yeast, so I can't comment on how easy it is to scale up YRC.

The real pain, both cost and time-wise, is the plating and screening - but I don't understand how YRC solves that. And I'm not sure I see where the big cost savings are coming from.

As far as building complex constructs, the other option is to assemble them in vitro using a polymerase-based assembly with synthesized oligos bridging the amplicons. Do you have any experience with how YRC compares with this?

Francisco - I completely agree about cheap synthesis making all of this infinitely easier, especially for labs that don't have solid in-house molecular biology. As a matter of fact, if you are just building one or two constructs it already makes more sense to just order them, given he the current price of $400/kb (though this is the lowest quote you are likely to find, and companies will often charge much more than their quoted price for "difficult-to-build" DNA). Academia hasn't realized this yet, but its already common practice in industrial biotech. However, we do need at least an order-of-magnitide reduction in cost before synthesis is cheap enough of large-scale library construction.


Alejandro Montenegro-Montero said...

Hey Nicolai,

Thanks for sharing your thoughts. While I agree with most of your argument, I have some comments:

I think that the time frames you mention are slightly wishful thinking. In practice, many of them will take longer, but as a general ballpark it's fine.

Also, I mentioned phenol cleanup, because I was trying to list the cheapest way possible to do the classic cloning (because YRC is cheap and I wanted it to be a fair comparison). The same thing applies to your comment on E-gels(expensive) and I didn't consider electroporation, as chemical transformation is cheaper (and takes longer).

Yeast transformation can indeed be easily up-scaled and the cool thing about it is that you can use your plasmid directly from the digestion reaction. No cleanups, dialysis, etc.

In our hands, cloning efficiency is over 90% (with 3k promoter fragments).

As far as building complex constructs, the other option is to assemble them in vitro using a polymerase-based assembly with synthesized oligos bridging the amplicons. Do you have any experience with how YRC compares with this?

In what sense? Cost-wise? I don't know, but it definitely involves more work (and money) that doing it in yeast.

Finally, I also agree that chemical synthesis can be the way to go in the near future.

As I commented in a previous comment, I'm now switching to bacterial recombinational cloning which will reduce cloning time even more! We now have the protocol and the bacterial strain needed and once I get some efficiency figures, I'll post about it, but I'm feeling very confident about it :-)

Nikolai said...

Electroporation is cheap if you make your own cells. Just buy a small stock of your favorite electroporation strain (I like NEB's 5-alpha), grow them up, and prep as much as you need. There are some decent protocols on OpenWetWare . It does take a full day though, and the later washes can be tricky, since the cells get less sticky and are therefore more easily lost when decanting. Having access to a cold room makes life much easier as it is critical that the cells stay at 4C.

Are you sure that in vitro assembly would be more difficult? I can't come up with any references off the top of my head, but I think the following assembly reaction should work:

1) Amplify your regions of interest using normal PCR.

2) For each pair of genes you want to be next to each other design one primer that will bind across the gap in one of the strands. Also design a pair of primers to amplify the ENTIRE assembly

3) Put in your amplicons and your primers and run a PCR.

I can't think of what would set the upper bound on the assembly size using this reaction, though I'm sure there is one. Since you can do all of your assembly steps in one reaction this might be easier than recombination. You might have a problem, though, if recombination has a size limit that your final assembly exceeds.

For more in vitro fun there is also sequence and ligation-independent cloning (SLIC): Li and Elledge, 2007, Nat Methods 4: 251 .


Nikolai said...

Though I just looked through the Colot 2006 paper and realized that recombination will do the assembly for you in a single in vivo step, which makes the in vitro reaction I talked about pointless. Is there a limit on the number of amplicons you can put together this way.

Alejandro Montenegro-Montero said...

Hi Nikolai,

Electroporation cuvettes are more expensive than making a CaCl2 solution :-). In Chile, grants are smaller and reagents cost 3 times more.

Are you sure that in vitro assembly would be more difficult?

It's not about being more "difficult" technically, but it will require the purchase of more primers and you'll be adding another level where things could go wrong or that may need tweaking (the ligating PCR, that is).

Since you can do all of your assembly steps in one reaction this might be easier than recombination

The assembly of the desired construct from multiple fragments can also be done in one "reaction" in YRC. You co-transform the different fragments into yeast along with the target plasmid.

For more in vitro fun there is also sequence and ligation-independent cloning (SLIC): Li and Elledge, 2007, Nat Methods 4: 251

Cool! Interesting paper. If you want to blog about it, our blog is open :-)

I want to emphasize again, that if I get bacterial recombinational cloning to work in my hands, it will be a better alternative than YCR, as you'll have your results overnight. Further, every lab can do bacterial transformation :-)

Francisco Barriga said...

Well, for the procedure Nikolai explained I think that the costs involved may be limiting for many labs. The other minor thing I´d like to comment is that fusion pcr is a very complex technique (in my hands I could only get it to work for up to 4 kbs).
In the case you want to build several constructs (or scaling up to 96 wells), the amount of primers needed for each particular reaction, plus setting the appropiate conditions for each and on top of that the cost of recombination enzymes I think that using bacterial or yeast-based recombination would still be a better option.

Regarding the upper limit for fusion PCR, that I am aware it has been described up to 20 kb (Nucleic Acids Res. 2004 Jan 22;32(2):e19)

For me the take-home message is this: it doesnt really matter which approach you use as long as it works(considering your lab´s resource limitations of course).

Alejandro Montenegro-Montero said...

Is there a limit on the number of amplicons you can put together this way?

We've tried 8 fragments (up to 1kb in size) with no problems. We haven't tried more or longer fragments, so I couldn't tell you what the limit is.