As a biophysicist in a biochemistry lab, I'm often learning new skills. To my surprise, recently I had to learn how to grow genetically modified bacteria to produce an artificial binding protein called an 'Affimer'. We use Affimers in our biosensor technology that I currently work on. Affimers are have the big advantage over antibodies in that Affimers can be made by genetically modifying bacteria and making the bacteria generate the Affimers for us. Making Affimers takes a few days of work with bacteria, whereas Antibodies, by contrast, take several months to make and require live animals (e.g. rats/mice/goats). As such, Affimers are much nicer to work with. For more info about Affimers see the website of Avacta, who sell them commercially.
So without further ado, here is my attempt at making a batch of Affimers, as told via the medium of live-tweets!
Step #1: genetic modification of the bacteria
I started off by retrieving some "competent" E. coli bacteria cells from the deep freeze. These "compotent" cells are useful in that they take up foreign DNA easily, and so can be genetically reprogrammed! I put the competent cells on ice for a while to let them thaw out, and keep them chilled. I then add a tiny volume of a DNA-modifying thing known as a 'plasmid' to the chilled bacteria.
Various colleagues at the University of Leeds have developed these plasmids over the past few years for the purpose of making Affimers. The plasmid contains DNA instructions that tell the bacteria how to make the Affimer protein that we want, and it also makes the bacteria resistance to the antibiotic carbenicillin, enabling us to screen genetically modified bacteria from regular old boring bacteria by subjecting the bacteria to antibiotic bombardment!
To introduce the plasmid to the bacteria, we transferring the bacteria from ice to a 50 °C water bath for 2 minutes before putting it back on ice again. The heat shock produces causes the bacteria's membrane to expand rapidly, enabling the plasmid DNA to sneak into the bacteria cells. The bacteria will then uptake the plasmid and integrate it into their DNA (don't quote me on the mechanics of this, I'm a physiscist).
With the plasmid introduction step complete, I have created genetically modified E. coli bacteria! All in all, the genetic modification bit was totally underwhelming - after all, people have been working for decades to make this stuff easy! 1/10 for drama, 10/10 for efficiency.
Step 2: growing lots of bacteria!
Next up in the process is growing lots of bacteria. To do this, you have to make a petri dish full of bacteria food and the antibiotic of your choice: we use carbenicillin. I had to mix up a bunch of bacteria food (yeast extract and other smelly stuff), and agarose gel (scientific jelly). This was all sterilized by heating it up in a helpful machine called an autoclave, which kills bacteria by a hefty dose of steam and heat. After the mix had cooled down to < 50 °C, I added a dose of the aforementioned carbenicillin antibiotics via a neat little syringe filter which filters out bacteria. This was all then poured out onto a petri dish and left to set into a jelly-like mixture.
I was quite excited because this was the first time I've ever used a petri dish! Again, I got some nice comments from the science community on twitter - they are great people!
I then got my genetically modified E. coli cells and smeared them all over the surface of the agar gel/bacteria food/antibiotic plate. I stuck them in an incubator at 37 °C overnight. and hoped for the best The theory is that the genetically modified bacteria should be resistant to the Carbenicillin antibiotic and any other bacteria - which aren't supposed to be there - will not grow.
I waited with baited breath overnight to find out that it had worked!
Those many tiny little specks on the plate surface are bacteria colonies, just starting out in life. So I scraped them up with a pipette tip (very scientific, much wow), and put them into a tube of bacteria food. This mix was incubated them in an orbital incubator at 37 °C around for a few more hours to allow the bacteria to grow up big and strong!
After a few hours the mixture had turned cloudy, with some gunk visible around the pipette tip I had used to transfer the bacteria to the tube. The cloudy appearance and snot-like gunk indicated that bacteria had grown more.
So next step was to grow the bacteria even more in a big flask full of bacteria food! To my amusement the big flask is quite like the stereotypical science beaker, which made me happy. The beaker and food had to be autoclaved to make sure they were sterile (tedious but necessary). To show you how transparent the food mixture was at first, I put it on top of my lab book, but you should NEVER EVER DO THIS BECAUSE IT IS A CONTAMINATION RISK! I then put this bacteria and food mix in an incubator at 37 °C for a few hours, in the hope that the bacteria will grow in what is known as 'The Log Phase' (that's the mathematical log, rather than the wooden variety), where lots of bacteria grow, but there aren't too many that they start to die off due to a lack of food and things like that.
As an aside: I noticed that one of my stock solutions had crystallized overnight. Fortunately I didn't have to use it again....
Also at this point, the fire alarm went off for no apparent reason. At least it was sunny!
STEP 3: MAKING THE BACTERIA PRODUCE MY AFFIMER FOR ME!
So the next step is where we force the bacteria to stop acting in their own best interests, and instead act in my best interest. After a few hours in the incubator the bacteria look like a badly poured pint of ale. At this point, the bacteria mix smelled really bad, and I'm glad I don't have to do this sort of thing often!
To this foul mixture, I added a chemical called IPTG, which basically tells the bacteria "make that protein we told you about earlier", to which the bacteria respond "yes master". Back in the incubator the mix goes for 6 hours, but this time at 25 ° C, to "encourage" the bacteria to express the Affimers instead of doing pesky things like growing and multiplying!
STEP 4: KILLING THE BACTERIA AND HARVESTING THE AFFIMER PROTEIN
To pass the 6 hours, I went to the cinema and saw 'King Arthur Legend of the Sword' because there was nothing else on. That passed the time quite nicely, even though it wasn't a particularly good movie. In fact, it was more like a weird cross between 'Dark Souls', 'Lord of the Rings', and a heist movie. I would not recommend unless you are really desperate for entertainment.
Upon returning to the lab I found that disaster may have struck because some unknown person had turned the incubator up to 37 °C instead of 25 °C! This was bad because at 37 °C bacteria focus on growing rather than making my Affimer protein! I was really worried that I would end up with little to no Affimer protein.
I was in too deep to quit though, so I pressed on. I poured the foul-smelling bacteria liquid into multiple tubes and spun them through a centrifuge like they were little bacteria astronauts. The bacteria did not pass this extreme-g test and ended up all smooshed together into a sticky snot-like gunk.
STEP 5: SEPARATING THE AFFIMERS FROM THE DEAD BACTERIA
To purify the Affimer proteins from the rest of the bacterial gunk, I added some lysis buffer to the gloopy stuff to break down the bacteria and some other chemicals to the bacterial enzymes from chewing up the Affimers. After about 20 minutes of that going on, I added the bacterial remains to some Nickle-NTA beads that have special his-tags which bind to the Affimers and only the Affimers. Essentially, I went fishing for the Affimer protein!
After a couple of hours, all the Affimers should be bound to the beads, so we discard the left-over crap, and add some elution buffer to unbind the Affimer from the beads.
After all that, the Affimers were separated from the beads, and I ended up with a sample of the purified Affimers. It's all rather underwhelming as there is very little to see - just a slightly opaque colourless liquid! To confirm that I had some Affimers, I put the sample into a spectrometer, which confirmed that I had made a few milligrams of Affimer protein. Yay!
I really should have made more Affimers, but problem with the incubator temperature really screwed up my batch. Nevertheless, I still made enough Affimers for a few weeks worth of experiments! Not bad for a first try....
Step 6: freezing the affimer samples
And of course the last thing is snap-freezing the Affimer samples in liquid Nitrogen for long-term storage! This bit is still a novelty for me and I love it!
So there we have it! That was my first attempt at growing bacteria for making Affimers. Despite all the hard work and bad smells, it was a weirdly enjoyable experience, but I would not like to do it often! Fortunately the University of Leeds have opened a protein production facility, which allows us to produce Affimers and other proteins to be made on an industrial scale - our first batch with this facility in June produced over 600 milligrams of Affimers in one go! Roughly 200 times more than my single batch! Now that's efficiency!
I hope you enjoyed this little blog post. Thanks for reading!