Troubleshooting a cloning experiment

One of the post-docs has been having a problem cloning the H. influenzae sxy gene into an E. coli plasmid vector. She needs the gene to be inserted in the ‘forward’ orientation but all her clones have it in the ‘reverse’ orientation. We’ve been operating on the assumption that this is because expression of sxy is toxic (we’ve lots of other evidence for this), but a bit of troubleshooting yesterday suggested that the explanation may just be a technical problem with the cloning.

The vector she’s using is one we obtained containing another insert, so she cut out the unwanted insert with the restriction enzyme SfiI, hoping to create a ‘no-insert’ version she could use to insert the sxy gene into. (I’ll explain the relevant properties of this enzyme below.) But the new SfiI ends of the no-insert version wouldn’t ligate together or couldn’t be recut after ligation (I forget which), so for her sxy cloning she instead just used the gel-purified vector fragment produced by the original SfiI digestion.

She designed SfiI restriction sites into the primers she used to amplify the sxy gene from H. influenzae chromosomal DNA, so alls he needed to do was digest the PCR product with SfiI, incubate it and her vector fragment with ligase, and transform the mixture into competent E. coli (selecting for the chloramphenicol resistance gene on the vector).

She got lots of colonies, but they all had the sxy insert in the wrong orientation. We now think this is because of a peculiarity of the SfiI enzyme. Wikipedia's explanation of how normal restriction enzymes work can be found here. SfiI’s recognition site is written as GGCCNNNNNGGCC; what’s peculiar is that it doesn’t about the sequence of the bases it cuts at (shown as NNNNN) – it only cares about the flanking GGCC bases. Typical restriction enzymes have no Ns in their recognition sites, so every cleavage site is the same, and because the sites are symmetrical the ends of the fragments have the same ‘sticky’ bases and can form base pairs that allow the ends to be ligated together. But the various SfiI sites have different bases at the N positions and, because the cut site is between the 4th and 5th N (moving 5’ to 3’) on each strand, ends generated from different cut sites can’t base pair and this can’t be efficiently ligated.

We now realize that the two SfiI sites flanking the original insert of the vector had different NNNNN sequences, and that this difference explains why the ends of the vector fragment couldn’t be ligated together (or why any rare plasmid resulting from ligation was no longer recognized by the enzyme). Furthermore, the SfiI sites on the PCR primers used to amplify sxy also had NNNNN sequences incompatible with the ends of the vector fragment. The details of the different NNNNN sequences are still unclear, so I’m not sure that this explains why inserts were obtained fairly efficiently but only in one direction.

It wouldn’t be unreasonable to be annoyed by discovering that we’d overlooked an important detail in our experimental design. But I'm always pleased when troubleshooting has discovered an error. I guess that I find it reassuring that we’ve been able to discover the reason why an experiment has been persistently not working. Of course finding one reason doesn’t mean there is no other problem with the experiment, but we’re optimistic.