Attempting to modify e-coli with CRISPR in my bathroom

20th of April 2018

I’ve been getting into biohacking. It’s slow going and there’s a fair bit to learn. However, there is a company called the odin who will sell you kits. They seem fairly straightforward — you can even order them from Amazon! So I figured I’d grab the e-coli and yeast kits and go from there. I thought I’d practice with the e-coli as modifying yeast seems a little more useful and interesting.

The process

  • Create a set of agar plates. Normal and anti-bacterial
  • Grow some bacteria
  • Take some bacteria and make it competent
  • Mix with transformation and the CRISPR and plasmids
  • Heat shock the bacteria so it takes up the plasmids
  • Place into the incubator with a bit of agar and leave it to grow a little
  • Spread onto normal and anti-bacterial plates and grow. See what happens

Biofridge

Since I’ve moved to the USA I needed to change out the plugs and what not. Turns out the transformer I used could cope with 110 volts, so all that was needed was to replace a few wire. I say all, I actually had a fair bit more to do because there were loose connections and mistakes here and there that I had to fix. With that done, I can now set the temperature I want and the fridge will attempt to meet it, turn off when it gets to temperature and back on if the temperature gets too low.

I made sure that everything was roughly calibrated up by comparing the readings on the box with two other thermometers.

Setting up the wet-lab

You need a few things to get going and the kit contains most of them. A very accurate pipette with tips, a rack to hold the small plastic vials, gloves, plates, plastic loops and a sharpie. Once I’d built the fridge, I figured I had enough to get going. Being in a bathroom helps. If I need to, I can always flush everything down the toilet!

Making plates and growing bacteria

When we want to grow the bacteria, we take the long, plastic sticks with a small loop at the end, and dip them into the e-coli vial, sort of like a bubble blower. You then scrape out the loop along the agar, streaking out the bacteria. Once that’s done, the lid goes back on the plate and is marked with date and time. A top tip I was given is to mark the bottom of the plate. Makes sense when you think about it — the lids can often be lost or swapped (apparently!)

27 degrees C seems like a good temperature to grow bacteria at. I left the plates over night and about 12–16 hours later, we’d gotten some good colonies.

Heat shocking

The guide suggests about 42 degrees C for heat shock and to use a water bath. At first I figured I’d just use the bio fridge as it can get to that temperature, but then I figured that using water probably has something to do with how fast the heat is transferred. So I decided to break out my k-type thermocouple and use a pan of boiling water on the stove. Once it cooled to the right temperature, in goes the vial!

Once the heatshocking is done, the bacteria goes back into the normal, cold fridge, to cool off for a bit, before it gets added to a smaller vial containing a little agar.

Fail

Looks like the bacteria didn’t grow in the end afterall. Not quite sure what went wrong but I have a few ideas:

  • Incorrect heat shocking
  • Contamination somehow
  • Incorrect temperatures
  • Non-viable DNA plasmids

I think I’ll carry on though. I still have my yeast kit left, which is a little more interesting. I figure I’ll try that next and try and tighten up my processes.

Freelance Research Software Engineer and Bioinformatics Student.

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