Hey folks,

I found a couple of papers that mentioned successful direct cloning into LAB, namely for plantarum and sake, even though most publications use a shuttle vector to assembly their construct first in E. coli.

So, I am currently trying to assemble a 2 insert 1 backbone construct via Gibson and directly electroporate it into different strains of LAB (eg. plantarum). But sadly I don't seem to get lucky -> no colonies.

I do know that the backbone works in those strains and I also verified the correct Gibson assembly via PCR. 

My question is, does anyone have any experiences with direct cloning into eg l. plantarum (or other LAB) and can share some insights? 

For example:

- How much DNA is used

- Change of electroporation conditions compared to normal plasmid transformation

- Clean up of Ligation or Gibson mix before electroporation

- What's the efficiency for you compared to normal plasmid electroporation

{...}

Thanks!