I have been currently currently cloning one gene into pTYB12 vector and we had troubles with it all the time.
Anyway, because we got no expression, I was currently trying to delete few first amino acids - putative vacuole targeting sequence. I've sent it to sequencing and the result at first glance didn't work. But when I checked once again, I found out, that the end fits to the plasmid (without the nucleotides as intended), but the first ~770 bp did not align. BLAST found 100% homology to E. coli genome sequences, but also to some weird plasmids, which had alignment at like 5 positions (again 100% identity, no gaps, no anything). When I checked on the internet, those plasmids are probably from E. coli isolated from nature. Some of the positions, where it aligned, were assigned as transposome elements or something like that.
It just seems weird, that it was incorporated just 2 bp downstream from the sequencing primer position, but I guess that's just a chance.
Anyway, my question is, whether has it ever happened to anybody of you, that your plasmid contained some transposome after cloning.
I haven't had it happen personally (that I know of) but I've definitely read about it happening, ex:
Article Reduced evolvability of Escherichia coli MDS42, an IS-less c...
If the region that it inserts into is toxic to E. coli then you'll get clonal expansion of the cells with the transposon insertion.
I also know it's happened with some of the larger sequencing projects years ago when they were using BAC libraries for sequencing, I think some of them actually wrote algorithms to find IS elements from the E. coli genome and discard them because it happened frequently enough.
I don't know which strain you used for cloning. It is most popular to use Rec minus (recombination mutation) strain to prevent recombination of unwanted DNA region like your example.
An expression strain is generally Rec plus (no mutation in recombinase or related genes).
So, if you used an expression strain to clone your insert, it is probable that recombination may have happened.
Yes I have certainly seen some examples of IS elements hopping into genes and stopping expression, generally I would expect there would need to be a strong selection against expression in order for such a mutant to be observable in the population, but it can happen. This is for the most part strain independent, so using a rec- strain won't have an effect on transposition.
Thank you for the replies (and sorry for delay, I was the last week off).
I used TOP10(F'?) cells. But it is possible, that it was already in the expression cells, because as I wrote, we had so many troubles with it already, that we attempted expression and when it failed, we isolated the plasmid and sent it to sequencing.
On a second thought, that one wasn't used for the final cloning though :-/
Anyway, thank you for your inputs. At least I'm glad I'm not the only one :)
- Badges
- Science topic
- Similar topics
- Biological Science
- Molecular Biology