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).
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.
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