complementing a double knockout in E. coli - (Mar/25/2011 )
I have managed to create a double knockout in E. coli using the lambda red system. Now I want to complement both genes in trans using the pBAD-TOPO TA expression system from Invitrogen (I've complemented single knockouts in the past with this vector).
Unfortunately I'm not very familiar with the details needed to express two genes under the single arabinose promoter in pBAD. Can anyone recommend resources for designing this?
I'm assuming I will need a linker sequence between the stop codon of the first gene, and the start codon of my second gene, but I have no idea. Any help would be appreciated.
i assume you are familiar with the architecture of the E. coli genome since you did a double knock-out?
you can look-up easily how operons in E. coli work (e.g. the lac operon). Normally, there is not much of a spacing region between two genes ...just a few base pairs and a RBS binding site in front of the second ATG.