bio.display

Today I performed the transformation (final) step with the BIO -RAD pGlo Bacterial Transformation Kit. This is the step when you actually put (transform) the DNA into the bacteria. The intended result is that you end up with a bacteria that has DNA it originally didn’t. In this case, it will be DNA taken from the jellyfish Aequorea victoria that makes it green fluorescent – that is, glow green when lit with a UV light.

Transformation doesn’t have a 100% success rate by far. Actually, only a few percent of the bacteria will be transformed. To get rid of the ‘normal’ ones, a simple trick is done: the desired DNA has an extra goodie build into it: it makes the bacteria resistant to an otherwise deadly antibiotic, called amplicillin. If the bacteria are put in an environment which is full of ampicillin, all the ‘normal’ guys will be killed on short notice, but all the ‘new kids’ will survive, and glow bright. To put it in SQL terms:

SELECT * FROM bacteria WHERE has_added_dna = TRUE;

The basic idea behind this step is that one creates all sorts of variations on the same theme, so as to see that really only those bacteria will glow that have the DNA inserted into them. In total, there will be four combinations of bacteria:

• +pGLO – ones that have the DNA added
  • LB/ampicillin – these will have the DNA in them, but they will not be expressed
  • LB/ampicillin/arabinose – these will have the DNA added, and they will be expressed (these will be the ones glowing)
• -pGLO – ones that don’t have theDNA added
  • LB/ampicillin – these will die all, as they don’t have the amicillin resistance from the added DNA
  • LB – this will grow to be regular bacteria

That is, if everything is done right.

Of course, it’s not enough to just add the DNA somewhere around the bacteria to make it enter it. After all, we’re all surrounded by lots of DNA all the time, and it doesn’t enter any of us. The trick is to shock the bacteria so hard, that the DNA enters it under the radar. Right after that, make it happy that it doesn’t really care, just tries to get back to normal fast – and will happily incorporate the new DNA into itself. And then we’re in.

In practice, this is done by doing the following.

First the DNA has to be prepared. It comes in plasmid form, which is like instant noodles – just add water, and you should be all set. (Well, sterile water, or our favorite transformation liquid in this case.)

The we prepare small test tubes to contain some of this transformation liquid, and put it on ice. (Cracked ice, like doing cocktails.) Two different tubes, actually: one marked -pGLO, the other +pGLO. (You’ll see the difference later.) We grab the bacteria that we’ve grown during the past day or two with a loop from the plate, and insert them into the test tubes, so that they dissolve in the liquid.

Now we reach for the dissolved DNA plasmid, and with new sterile loops, insert some of the stuff into the test tube marked +pGLO. (Guess you figure by now, the one marked -pGLO don’t get any of the funky DNA.)

But back on ice, and leave it cool for 10 minutes.

Then comes the heat shock: get the bacteria from the ice (which is 0 °C), and put unto a heated pad of 42 °C for 50 seconds. Like a good sauna, they’re gonna trying to catch their breath as well. As the time is up, but back on ice for another 2 minutes.

Now that they are in shock and asking: “What the hell is going on?”, we’re trying to comfort them by at first giving them a lot of food. This is done by inserting a lot of LB nutrient into the test tubes, all of them. (This is the same LB that is found in the agar on the plates.)

The second part of making them comfortable is to put them into a nice climate. For that, they need to be put on a plate – of which we’ll have four different in total (see also above):

• +pGLO / LB / ampicillin
• +pGLO / LB / ampicillin / arabinose
• -pGLO / LB / ampicillin
• -pGLO / LB

This is done by new sterile pipettes, put a drop (100μl) of the bacteria mixture on the plate, and spread around with a sterile loop evenly.

As this is done, we finish the comforting procedure by putting them into an incubator of 37 °C, where they will feel like being on the beach having cocktails.

Hopefully they will grow by tomorrow, as intended. Unless, of course, if I made mistakes, which I have. To be on the safe side, I made the experiment with 4 different sets, named after the color of the test tubes they were in: green, yellow, orange and purple. I guess this was a good approach, as the mistake I already know I made are:

• I didn’t prepare the pGLO DNA plasmid a day in advance, as the manual said (ouch – this has an effect on all experiment sets)
• I heat-shocked the yellow set twice
• not sure if followed the sterile guidelines always

Well, we’ll see soon if they still got through though 🙂