We have been struggling for a couple of weeks to get one gene fragment (for RNAi) cloned into a plasmid. Normally this plasmid (with and without insert) grows perfectly in E. coli and we cloned other gene fragments in parallel and everything worked, we have them in planta now.
Here’s an overview to the very simple cloning experiment (which we repeated already with same results…): after cloning our vector containing our ‘problematic’ PCR product, the yield was only two E.coli colonies- we confirmed the presence of the gene insert by doing colony PCR and PCR after plasmid purification (we had the correct fragment size on gel). We sent the plasmid to be sequenced to re-confirm the identity of our inserted sequence, and surprise! We only obtain plasmid sequence, meaning the colonies contained empty vector only- we restricted the plasmid with the enzymes used for cloning and no insert was found. And the same result after two rounds of cloning. We were quite irritated, as the PCRs with the primers that are contain cloning site + gene specific sequence always worked, indicating the presence of our insert in both the colonies and after plasmid purification..
I went to check literature for the possibility of this gene fragment being ‘unclonable’, and came across the PanDaTox database (http://exploration.weizmann.ac.il/pandatox/1_0/home.html). After blasting a couple of gene fragments that previously worked to the database, and my ‘problematic gene fragment’, it turns out the only with a very low e-value (and therefore high probability) of ‘plasmid unclonability’ is the ‘problematic’ one.
My general question: if anyone has come across these unclonable gene (or fragments), how efficient is it to use in silico tools (like PanDaTox ) to predict the (un)clonability of a gene?
A more specific question: I don’t really understand how a small gene fragment can be ‘toxic’ to E. coli- my insert is 200bp in size, and only the last 32 bp (3’ on the gene) are identical to a ‘toxic’ gene- this appears to be enough for it to be unclonable (from the experiment). How does this toxicity work? I appreciate in advance any suggestion.
This is how I plan to proceed: I will use another sequence, further upstream of the same gene, since PanDaTox predicts a better clonability. I am planning to repeat the experiment using new primers to hit this gene fragment instead… and hopefully get better results.
Thanks for taking the time to read this and in advance for any answers.
Perhaps the problem isn't protein expression from your gene fragment- does the sequence have low or high GC content, or a lot of secondary structure? Those could be other reasons for the cloning problem. You could also look into IDT minigenes- when you add your sequence online the IDT tool will tell you if there is a potential problem with the sequence, and they will refuse to try if it is particularly difficult.
Thanks for your time to reply Jennifer. The unclonable sequence has indeed a complex secondary structure (and low GC content), but this is also the case with other gene fragments we have cloned with success previously- that's why I was hinting at toxicity. IDT tool seems a useful tool, I was using Geneious for this purpose.
The issue of "unclonability" here is somewhat controversial - you actually cloned it (as shown by colony and plasmid PCR). It seems to me, your colony was not a single clone - probably mixture of empty vector and correct insert, where the correct one represents just negligible minority - it is enough for PCR (with one insert specific primer), however sequencing (probably using vector specific primer) preferred the empty vector.
You can sequence your correct PCR product rather than purified plasmid. If this is ok, you need to "separate" correct and wrong construct. Maybe, there is a unique restriction site in the empty vector, which is absent in the construct - digest the mixture and re-transform it.
Still, there is an issue - is your sequence toxic or unstable? As you mentioned, toxicity of such small fragment is not very likely. But it can be unstable (tends to recombinate etc), especially if it is a tricky sequence (hairpins, repetitions...). Try to grow your bacteria at lower temperature (or use a stable E. coli - like NS, Stbl2).
In theory, you can try to monitor the undesired selection pressure (disappearance of your correct clone) by qPCR. Just inoculate selective medium with leftovers from your transformation and run quantitative PCR at different times of cultivation. You can monitor your insert sequence and the vector alone (using primers, not spanning MCS). Their ratio should be roughly stable, if there is no toxicity/unstability. If your construct is toxic/unstable, this ratio shoul rapidly increase (empty vector should prevail). I would not start with this, but can help at the advanced stage of frustration.
Good luck.
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