Plasmid Insertion

Getting bacteria to take up plasmid DNA on command is a by-the-numbers process, You’re going to kill many of your intended targets, making this a great argument for only doing genetic modification in microorganisms until we develop delivery mechanisms that don’t involve so much… death. When performing these protocols, 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. You’re then going to kill every bacteria that didn’t take up the plasmid using antibiotic selection.

There will be survivors… but at what cost?

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)

However you choose to prepare the cells, the plasmid insertion step remains the same. Heat shock is an incredibly quick and easy protocol, most of the hard work is in preparing the cells;

Protocol: Heat Shock Transformation of chemically competent E. Coli

2. Electroporation

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. I am yet to try them myself so that I can add in tips to reduce reagent costs or replace the electroporator with a DIY alternative. As always, a huge thank you to Coleman Labs for the protocols;

Protocol: Preparation of electrocompetent cells of Pseudomonas or E. coli

Protocol: Electroporation of electrocompetent cells of Pseudomonas or E. coli

3. DIY Gene Gun

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.