3 out of 4 ain’t bad
(It’s better than Meat Loaf’s 2 out of 3.) This week I attempted to clone 4 modified versions of the ICAP CRP-binding site. In a previous post I described my attempt to use site-directed mutagenesis to change ICAP. That approach failed, so I returned to the method used to originally clone ICAP: complementary synthetic oligonucleotides containing the ICAP sequence (or variant sequences) were allowed to anneal and the resulting dsDNA 28 bp molecules were cloned.
As described in the earlier blog post, the ICAP palindrome poses a serious problem because of the formation of very strong hairpins, which directly competes with inter-molecular annealing. Consequently, cloning synthetic oligos has a very low efficiency (I was lucky with ICAP; the single positive clone had the ICAP sequence!), and this is why I had opted to try site-directed mutagenesis. This time around, oligos were melted at 95º and then given 10 hrs to cool to room temperature, an approach that should hold oligos at high temperatures where inter-molecular bonds are stable and hairpins are not.
This round of cloning was slightly more successful. Each ICAP variant had at least 5 positive clones (ie. white colonies in blue/white screening on X-gal) except for ICAP-9G,14C (2 white colonies), which has the strongest hairpin structure of all ICAP variants.
My sequencing results came in on Friday and all but ICAP-9G,20C came back positive as having the desired sequences. Unfortunately, there was no sequencing read for ICAP-9G,20C, thus I may in fact have the desired clone, I just don’t know it. I will talk to NAPS on Monday about what may have gone wrong. The strong ICAP secondary structure had been a problem in earlier sequencing reactions, but this appears to have been circumvented by addition of betaine to sequencing reactions. Even if the ICAP-9G,20C secondary structure remains problematic during sequencing, the reaction should have at least yielded sequence up to the hairpin; the “no sequence read” is a mystery, so I really hope the folks at NAPS have a good idea of what to try next. This weekend I extracted plasmid from the other ICAP-9G,20C positive clone, so I’ll take that in for sequencing on Monday as well. Given the success of the other three clones, I remain confident that ICAP-9G,20C will also turn out well.