Getting E. coli to take up plasmid DNA when we want them to is a by-the-numbers process and a great argument for only doing genetic modification in microorganisms until we develop delivery mechanisms that don’t kill so many of our intended targets. In short you’re hoping to compromise the integrity of the cell wall and membrane enough to let the plasmid sneak in, but not enough to kill the bacteria entirely.
1. Heat Shock Transformation
In order to use the heat shock transformation protocol, we must make our cells “Chemically-competent”. If you’re lucky enough to have access to a -80°C freezer then you will only need to do this protocol once and can store a huge quantity of cells for future projects. Storage at -20°C will dramatically decrease your transformation efficiency.
Protocol: Preparation of chemically competent E.coli cells (rubidium chloride)
This protocol gives excellent transformation efficiency, but RuCl is extremely expensive. As such we tend to use the next protocol.
Protocol: Preparation of chemically competent E.coli cells (calcium chloride)
Protocol: Heat Shock Transformation of chemically-competent E. Coli
Electroporation is a technique that can help you get good transformation efficiency in a wider variety of strains than Heat shock, but requires a dedicated piece of machinery known as an “Electroporator”.
These protocols are a direct transcript from the Coleman Lab protocols. We are yet to try them ourselves so that we can add in tips to reduce reagent costs or replace the electroporator with a DIY alternative. As always, a huge thank you to Nick and his team at USYD for supporting our lab.
Protocol: Preparation of electrocompetent cells of Pseudomonas or E. coli
Protocol: Electroporation of electrocompetent cells of Pseudomonas or E. coli
I just had to include this here, I haven’t tried it yet – but maybe the idea appeals to you? I’ve always wanted to fire my DNA into my bacteria at extremely high velocity using what is effectively a pneumatic cannon. It’s on the to do list I promise.