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A miniature light show in the palm of your hand

I posted about western blots and protein gels recently, but they’re actually more like a validation for some big experiments I did last week. These are the big experiments I was referring to in some previous posts. I needed some Coxiella strains that make different proteins for me (I’ve posted quite a bit on this), so that I could use them to infect some human cells.

So, when I say human cells, they’re actually cervical carinoma/cancer cells. We use cancer cells all the time in the lab because, well, they’re very hardy. Cells can divide and replicate, but every cell has a limit on how many times they can do this. Most cells replicate a few times before they close the curtain, but cancer cells… they’re practically immortal. These cells in particular are from a woman named Henrietta Lacks. There’s a whole story on how she never gave permission for her cells to be harvested, but now these cells can be found worldwide. They’re actually one of the most commonly used epithelial cell lines (epithelium is the outer layer of tissue, so these cells form a ‘skin’ like barrier around different structures) in the world.

Anyway, we have some HeLa cells (that’s what these cells are called), and we grow them in liquid culture media called DMEM (Dulbecco’s modified Eagle’s medium- weird name), with a bit of serum to boost the nutrient levels.

Image result for dmem
A typical bottle of DMEM that we use to grow our HeLa cells.

HeLa cells are epithelial cells, meaning they self-adhere to the surface of their container. When we grow them, they grow on the bottom surface of our flasks, our plates, etc. It takes about 8 hours at 37 degrees, 5% CO2 for them to gentle settle and attach.

Image result for mammalian tissue culture
A typical tissue culture incubator looks something like this.

So for my experiments, I can get these cells, count them, and ‘seed’ them into an assay plate that looks something like this:

A typical 96 well black assay plate

We set up conditions in triplicate, so there’s three wells assigned for each condition in an experiment. If you look at it from the top, you’ll see wells that are pink, that have cells/samples in them, and clear wells that don’t have anything in them. You have 96 wells, so you can test many different conditions on the one plate.

Image result for 96 well assay plate with media
Here’s an example with a clear, see through plate. You can add cells quickly by using a multi-channel pipette like the one in the image.

Once I add my cells and leave them to incubate and self-attach, I can add my relevant Coxiella strains. Some strains make an artificial protein that I’m basically forcing the bacteria to make, so that I can study its activity. One portion of the protein is a Coxiella one, but the front portion of it is a foreign protein from another bacterial species (completely unrelated to Coxiella).

The red and blue in these ‘fusion’ proteins are from Coxiella, but the yellow isn’t.

The yellow portion of these proteins is actually a bacterial enzyme that cuts/cleaves things called ‘beta-lactam’ rings. If you know your antibiotics, you may have heard of beta-lactam drugs. They’re your penicillins, amoxycillins, ampicillins… anything that ends in ‘cillin’, really. The yellow protein is called beta-lactamase, and anything that ends in ‘ase’ is a protein enzyme that can modify its target in someway. In this instance, beta-lactamase can cleave beta-lactam rings found in certain compounds. It gives rise to antibiotic resistances in certain bacteria, and makes beta-lactam drugs completely useless against them.

So why am I playing with beta-lactamase? Well, I’m not working on antibiotics, for starters, but we can use this enzyme to our advantage.

There’s a kit that we use, and this kit comes with a special chemical compound. This compound, called CCF2-AM, contains a beta-lactam ring. Under normal circumstances, when you bombard this compound with a specific wavelength of light (410 nanometres), it emits a green colour (or 520 nanometre wavelength of light). But! In the presence of beta-lactamase, this enzyme can cleave the beta-lactam ring in this CCF2-AM, and the structure changes (because it gets cut almost in half). When CCF2-AM is cleaved by beta-lactamase, the light emitted turns into blue (or 450 nanometres). This colour difference is key.

The big honey comb looking structure is the CCF2-AM compound. Beta-lactamase can basically cleave this compound and this changes the colour of light it emits.
Newton P et. al. 2016. J Vis Exp

Lets add another layer of complexity (because, why not?)

I’ve got these infected HeLa cells. They contain lots of Coxiella. Healthy, normal Coxiella have this really cool mechanism of injecting (‘translocating’) bacterial proteins into the host cell, essentially by stabbing through membranes.

It’s that structure in this diagram that’s labelled, T4SS, which stands for Type 4 secretion system. You can see how during replication inside cells, these structures can pierce membranes and inject bacteria-derived proteins into the host cell.

The crux of this experiment is to figure out whether a particular protein of interest (the red or blue protein in the ‘Plasmids’ diagram) is getting translocated (injected) through the T4SS, into the host cell.

Because I’ve physically fused my proteins of interest with beta-lactamase/yellow protein, if the protein gets translocated through the T4SS, then beta-lactamase will also be found in the host cell (outside of the bacterial cell).

SO (still with me?)- putting all of this together:

Beta-lactamase, as I said, can cleave CCF2-AM. Uncleaved CCF2-AM emits a green colour, but cleaved CCF2-AM emits blue. Cleavage/blue signal can only occur if beta-lactamse is present in the host cell, outside of the bacteria.

So what I can do is basically add CCF2-AM dye to my infections, wait a couple hours (for the dye to be absorbed by the HeLa cells and get cleaved/not cleaved by any beta-lactamase present), and then put it in a special machine and read how much blue vs green signal is getting emitted by the cells.

Image result for clariostar
The machines look like this. You pop the plate in and it will bombard the samples with whatever wavelength of light you set it to (in our case it’ll be 410 nanometres), and it will detect and quantify how much green (520 nanometres) and blue light (450 nanometres) is being emitted at the same time. Pretty nifty!

All in all it looks something like this

Technically the T4SS should be coming out of the longitudinal ends of Coxiella.
Newton P et. al. 2016. J Vis Exp

CBU0077/MceA is a specific Coxiella protein we know is translocated via the T4SS. When fused to beta-lactamase (BlaM), and is being produced by Coxiella, it will translocate out into the host cell, dragging BlaM with it. Out in the host cell, the BlaM enzyme will cleave any CCF2-AM present, and cause a shift in colour emission so that there’s more blue over green. The machine we use to read the plates spits out numbers, and we can calculate them to get a blue over green (450:520 nanometre) ratio.

You can look at it visually, too (rather than just as numbers).

You can count how many cells there are in your field of vision/image, then calculate what percentage of those are blue. The little ‘p’ means ‘Plasmid’.
Carey and Newton et. al. 2011. PLoS Pathog

The specific experiments I was doing last week used this technique, but at a more complex level involving infecting the same HeLa cell with different strains of Coxiella at the same time. It’s a little too hard to explain right now so I’ll leave it at that!

Hopefully this made some sense- it’s more just to illustrate how many layers an experiment can have (human cells, bacterial cells, plasmids, proteins, chemicals, light…), and how we can use natural systems (antimicrobial resistance) to our advantage.

Categories: General Lab Ph D posts

Tagged as:

ABugsLife

A former wet-lab based Bacteriology Ph. D student residing in Australia. Now working part time at a secret location as a Communications and Data Officer. 👀 🦠 🧫 🧬

6 replies

  1. OK Miku. I didn’t understand but I can see the cleverness of it all – the amazing way it can all be interpreted. But in the end, what is it for? What problem that is affecting us is it going to help to fix?

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      1. Or more or less, if we understand what it’s doing inside the cell, and how the cell responds to it, we can find out more about how they both work. Someone else might find it interesting and identify ways to weaponise it against the pathogen, or maybe against human cells that have gone rogue.

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