RNAi induction kills my bacteria?!

Model Organism: Worms Methods & Reagents
1

Hi everyone,

I tried making my own RNAi clone for the first time (by cloning a ~800 bp piece of my gene of interest into L4440 and transforming into HT115 E. coli). The RNAi clone grew fine in LB/Ampicillin when I grew the overnight culture, BUT when I put the bacteria on RNAi plates containing IPTG and selection antibiotics the bacteria died. Is it possible that the dsRNA expression somehow killed the bacteria? If so, what can I do to present this - i.e. how should I redesign my RNAi cloning? I already checked to make sure I hadn’t disrupted the antibiotic resistance genes during my cloning, and in fact I didn’t (I only used rrestriction sites in the MCS). I have used many Julie Ahringer library RNAi clones without ever experiencing this problem, and in fact my control RNAi and other RNAi clones grew fine on my RNAi plates this time too!

Thank you very much!

2

It seems doubtful the dsRNA killed the bacteria, unless one strand encodes a toxic protein.

Are you growing the HT115 on both tet and amp? Make sure your host HT115 competent cells are still tet-resistant.
Although not needed for maintenance of a pPD129.36 ‘feeder’ plasmid, the tet maintains the DE3 lysogen that
contains the lacP-T7RNApol (IPTG-inducible) fusion gene. In the absence of selection on tet + amp, the strain could
lose DE3, which would result in both tet-sensitivity and a lack of dsRNA accumulation following IPTG treatment.