Tag: Biology

What’s a PCR and how does it work?

In the last article I ended having a DNA template thanks to Prof. Yu Fu and I had a DNA sequence of the region where I want to integrate my DNA construct. So to now I need to copy a piece of the DNA plasmid (ring) that I have and add a stretch of DNA sequence (~ 50 bases) that are complementary to to the point in the yeast genome where I want to integrate that peace. I can do both in one step with a technique called Polymerase Chain Reaction or PCR.

A PCR is a method to produce DNA without using living cells to do it. It is something like a molecular copy machine. The main ingredients for a PCR are an enzyme called DNA-polymerase, which does the copying, dNTPs, which are the building blocks of DNA, the template DNA from which I want to copy and primers. Primers are small DNA peaces (normally about 20-25 bases long) that define the DNA stretch I want to copy. The forward primer (the start signal) is complementary to the sequence where I want to start the copy and the reverse primer is identical to the sequence where I want to stop the copy.

Schematic drawing of a complete PCR cycle
Schematic mechanism of PCR. CC BY-SA 4.0

The picture above is from the Wikipedia article on PCR that also explains it really well in detail. As you see the first step of a PCR is making the DNA accessible for the copying by heating it until the typical double-strand structure splits up into two single strands. For this you heat it to something around 95 °C. Then you cool it down to the annealing temperature which depends on your primers but is normally a bit above 50 °C. To start the DNA-polymerase you now heat it again to something around 72 °C. This sounds like a very high temperature for an enzyme and it is. The trick is that the polymerase comes from the bacterium Thermophilus Aquaticus (or others) that lives around hydrothermal vents that spew water, smoke, acid and other stuff at temperatures of more than 100 °C. So their enzymes are optimized to work in extremely hot and unpleasant conditions.

Now you repeat these three steps over and over again and with each repeat the number of your DNA constructs doubles. This means you have an exponential growth from 1 to 2 to 4 to 8… to a bit more than a million after 20 cycles to a bit more than a billion after 30 cycles. So you create a lot of identical DNA in quite a short time (normally 1 to 4 hours).

The machine that adjusts the temperature in a programmed manner is called a Thermocycler and looks quite unspectacular.

A typical laboratory Thermocycler made by Eppendorf, own work

This explains how I copy the stretch of DNA from the plasmid but I also wanted to add some sequence to the ends. That’s actually quite easy. I just add the sequence to the primers because they are the beginning and the end of my newly created DNA constructs. Now you might ask: And how do you get these primers? Well like most things today. I order them online. There are companies specialized in producing custom DNA. Basically I could also save myself the whole work and just order my whole construct online but that is still quite expensive. While my exceptionally long primers (primer + sequence are about 60 bases long) cost something between 20 and 50 $, my whole 2500 bases long construct would cost about 1.000 $ to order. That is still quite a difference but it gets cheaper and in a few years biologists will probably order a lot more DNA than they create using molecular techniques like PCR and cloning.

Now that I have my PCR product I want to verify if it is the right one. The easiest way to do that is to put it on a gel that lets short DNA pass quicker than long DNA. Than you add voltage and the negatively charged DNA wanders towards the positive electrode and splits up by size. If you add a marker that includes DNA pieces of known size and a compound that makes DNA visible under UV-light (ethidium bromide) you can see what size the fragments that you produced have. That normally gives you a good idea if the experiment ran as expected.

I did all of that and on the first try the result was no visible DNA at all. Than I changed a few parameters and tried again and saw a very thin band where I expected it and a few others where they shouldn’t be. So I changed some more parameters and on the third try I got this wonderful picture of a gel with a strong band at exactly the position where I expected it.

This made me quite happy but now I had to do something with it. I wanted to put this DNA into a yeast cell and make it integrate into the genome. This is called transformation and is quite tricky as well. I’ll explain that the next time.

Teaching Yeast to Age like normal People Part III

Yeast cells growing on a selective plate, own work

In the post “Teaching Yeast to Age Part II” I explained what I tried first to teach my yeast to shorten its Telomers at each cell division. I also told you that it did not work yet. While I have not yet completely given up on Tretrad dissection, I decided to first try another way. It would already be nice to have my fluorescently tagged cells with a knocked out Telomerase but it would be a lot cooler to be able to switch the Telomerase on and off at will.

power on and off switch on wall
Photo by Mikhail Nilov on Pexels.com

The way I’m trying to achieve this is called promoter shuffle. As you might know, Proteins (like the subunit of Telomerase I’m interested in) are encoded in the genome by a fixed code where 3 letters (called a codon) in the DNA (bases) are translated into one letter in the protein (amino acid). This code is well known and understood. All codes for proteins start with the same start codon and end with one of three stop codons. However to really produce this protein in the cell there needs to be more. The gene needs to be flanked by regulatory code that influences if and how strongly the encoded protein is produced. Most important for this is the so called promoter region. This is the code directly before the start codon. It is normally between 50 and a few hundred bases long. Their code is a lot less understood than that for the protein. There are strong and weak promoters, promoters that are always active and promoters that are switched off or on on specific stimuli.

Since I want to make the Telomerase switchable I take the genetic code for one of its subunits and exchange some of the code before its start codon against something else. There are a handful of common switchable promoters in yeast that are well described but most of them have some disadvantages. I identified a relatively newly identified one that I hope will be a good choice.

It is the promoter of the gene DDI2 that is normally almost inactive but increases its activity more than 1000-fold if the cells are incubated with the chemical cyanamide. In the concentration it is used here cyanamide has almost no effect on the yeast cells.

Exchanging code in the yeast genome luckily isn’t too complicated. Yeast quite readily incorporates genetic material that you feed it if a stretch at the beginning and end of the inserted code match the code in the genome. This process is called recombination. Recombination is also the process that makes children unique instead of copies of their parents because it randomly mixes the genetic material of your parents that is passed on to your kids.

So I need a stretch of DNA containing the 500 bases before the start codon of DDI2 with 50 bases on both ends from the area before the start codon of the Telomerase subunit. I did a lot of thinking how to build this myself but it would have been a lot of work. Then I thought about the paper where I first read about the DDI2 promoter. In the nature article “Fine-tuning the expression of target genes using a DDI2 promoter gene switch in budding yeast” Professor Yu Fu of the Chinese Academy of Sciences and colleagues characterized the promoter and explained how they integrated it in genomic locations.

So I just wrote an email to Professor Fu asking if I could get some of the material they used. He actually replied within a day and said that he’d be happy to help me. He sent me the code of the promoter without expecting anything in return. That’s a great thing about the science community. Most people are happy to share and are really nice if you show interest in their work.

So all I have to do now is add the code stretches at the ends that determine the target where I want to integrate it. I’m doing this with a PCR which I’ll explain in my next article.

Teaching Yeast to Age Part II

In my last post I explained how to get a bag of 4 yeast spores of which one hopefully has the two mutations I want in my new yeast strain. Now comes the really tricky part: You have to find this spore.

Yeast cells under 1000x magnification (DIC), own work

Before I explain the method you have to get an idea on how small one yeast cell is. The average yeast cell is about 3 to 4 micrometers. The smallest distance between two dots to still see them as two with the naked eye is 0.1 mm which is about 300 times a yeast cell. A human hair is on average between 0.06 an 0.08 mm thick, which is 20 times a yeast cell. If you look at the picture above, depicting yeast cells magnified 1000x, the whole frame is about the thickness of a hair.

OK, so they are really small and not visible with the naked eye but we want to find a single spore (that is again smaller than a normal cell) and identify it. The method we use for that is called micromanipulation. A micromanipulator is basically a microscope with a stage (on which you fix the plate with your cells) that can be moved in tiny increments. And it has a lever with a very thin glas needle fixed to it that through some mechanics can also be moved in a very small scale.

My lab has quite a cool and modern micromipulator that has a joystick to move the stage and a computer driving you to positions on a virtual grid on your plate where you can put single cells.
What you now do is you look through a patch on your plate where you’ve put some of your cells. Once you’ve found a bag with 4 spores (which is called a Tetrad) you move the tiny needle really close to it. A moment before the needle would actually touch the plate a meniscus of water forms between the needle tip and the plate that scoops up the Tetrad and hopefully nothing else. Then you move to an empty position on the grid and, by moving and shaking the needle while it almost touches the surface, you try to deposit one of those 4 spores. Then you go to the next spot and do that again. So in the end you get a plate full of 4 spots in a row where you deposited single spores. You can see how it works in the following video.

Me showing how a Tetrad dissection works.

After finishing the sorting I put the plate to 30 °C for one or two days to give the cells time to grow. The result looks like this:

Dissection plate with colonies on it, own work

As you see not all cells I’ve laid out have grown. That is quite normal because not all spores are viable after the procedure. On the left side was the reservoir, where I’ve put a lot of cells to look for Tetrads. I’ve cut that off with a clean scalpel so the cells do not overgrow the rest of the plate.

In the next step now I have to identify which of these cells are really derived from spores (and are haploid) and which of them are actually just normal diploid cells that accidentally lay around with 3 buddies and looked like Tetrads. To do this you use another nice feature of yeast. Wild yeast can live of a lt of things and can make everything it needs (like amino-acids and bases for DNA) semselves. The labstrains you normally use however have some genes they need for this knocked-out. This means to let them grow you need to add some amino-acids and other things to the medium. Now the trick is, that the genetic traits that I want to breed together here are marked with the genetic code that the laboratory strain needs to make these amio-acids themselves. That means I take this plate and stamp it over to a plate where the medium is missing two special amino-acids, only those cells can grow that have both genetic traits that I need. This is called selection.

The problem here is that the diploid cells I mistook for Tetrads also have both markers and can also grow. Here however the statistic can help. Since I always put all 4 cells that were together in one row and in a Tetrad only one of those 4 can have both of the genetic markers, I now have to check my selection plate for rows where only one of the 4 cells has grown. Another test that I can do afterwards is to see if the cells of such a colony mate with other haploid cells of either mating type. Diploid cells won’t do that.

Original plate on the right, selection plate on the left, own work

The results look relatively promising but further analysis showed that this actually hasn’t worked. However this is an important part of science: Things don’t work. After trying this a couple of times I decided to first try another way to teach my yeast cells to age that is a bit more elegant and hopefully works better: I will put a switch on the gene for Telomerase so I can turn it on and off as I want. I’ll explain how this works the next time.

Finding the fountain of youth and curing cancer

boy standing on outdoor fountain
Photo by Rene Asmussen on Pexels.com

Well if that’s not one of the click baitiest titles you’ve ever seen. But that’s what I hope being part of with my PhD project.

In the last article I explained what Telomeres are and why they are important as one of the possible keys to aging on one and cancer on the other side. If you haven’t read the article yet, you can find it here.

When it comes to Telomeres it’s all about their length and its changes over time. If Telomeres degrade it is a sign of cellular stress (in some cases the opposite is true) or strong proliferation and if they reach a certain critical length they stop the cell from division and send it into senescence. If a cancer cell wants to divide further it has to overcome this brink and find a way to elongate its Telomeres.

This shows you why drugs that influence Telomere length (in either way) have a huge potential. But how do you find these drugs? There are several answers to this question however often the answer is high throughput screening. Which is a nice word for trial and error.

In high throughput screens you develope a system that gives you an easy, reproducible and cheap read-out for the effect you hope for and then you make tens or even hundreds of thousands of experiments in parallel with huge libraries of chemical compounds. If some of them show a promising effect you try to reproduce that effect first in the same and later in other experimental setups. Then you try to find out how your candidate compounds work, if they are toxic and other questions before you can start developing a drug from them.

For Telomere length there is currently already a problem in the first step. There is currently not yet a really easy, cheap, quick way to measure Telomere length in high throughput assays. For an overview on the currently available options you can read this review paper by Lai et al. 2011

Proteins associated with the Telomere, Picture from Vaiserman and Krasnienkov. 2021

My idea seems to be an easy (if probably not very accurate) option. Telomeres are normally quite tightly packed with proteins. I want to use this Telosome as a molecular ruler. My idea is to optically tag one or more of these proteins and look for a correlation between an optical read out from these tags and the length of the Telomeres. If I find this correlation I only have to calibrate it and I’m ready to screen for the pill against aging or cancer.

In the first step I’m using the bakers yeast Saccharomyces Cerevisiae as a model. I’ll explain a little more about the advantages and disadvantages of this system in another article. For now one of the advantages is that my lab has a library of yeast strains with tagged proteins. These tagged proteins are fusion proteins that were made by adding the genetic code code for Green Fluorescent Protein (GFP) to end of the code for (basically all) yeast proteins.

So in the first step I’m just taking yeast stains out of the – 80 °C freezer and revive them. Now I only have to check the fluorescence signal from cells with different Telomere lengths and see if there is a difference.

It could be so easy but there’s a problem with yeast. It doesn’t age or at least it keeps it’s Telomeres very constant in length using Telomerase. So I first have to teach my yeast to age like our somatic cells. I do this by disabling their Telomerase. I thought about three different methods of doing this that I’m currently working on but I’ll explain them in other articles.

On the other hand I can not only make their Telomeres shorter but also longer. This is interestingly done by cultivating the cells in 5 % alcohol.

If I don’t see a correlation in this easy system I have a few more elegant ideas that I’ll explain in other articles.

A short story about aging

Why do we age? What seems like a biological necessity is actually not. Single cell organisms in most cases could be called immortal although the definition is somewhat problematic since for most it is hard to distinguish between mother and daughter, between individual and a population of clones.

Turritopsis dohrnii medusa
Turritopsis dohrnii medusa (CC by Bachware)

There are however also more complex life forms that are not aging as we do. The only really biological immortal animal we know of is the jellyfish Turritopsis dohrnii. It can rejuvenate itself by re-entering the polyp stage and re-emerging as a jellyfish. But there are also mammals that defy our traditional understanding of aging: The naked mole rat.

Naked Mole Rat
Naked mole rat in Zoo (CC by Roman Klementschitz, Wien)

The naked mole rat is a funny animal through and through. They look like sausages with teeth that could use a dentist. They live underground in insect like states where only the queen is able to breed. Becoming the queen normally means beating the current queen in a bloody usurpation. They have an amazing immune system that basically completely protects them from illness, cancer and infection. And they do not age like we do.

Aging for us is mostly defined by a decrease in physical fitness, the slow loss of function of our bodies after the end of the reproductive phase. It is also an increase in mortality rate (the probability to die within a fixed amount of time). Naked mole rats however show neither an end to reproduction, a decrease in health or fitness nor an increase in mortality with age. That doesn’t mean that mole rats are immortal. On average they become 15 years old but that is mostly because of injuries from fights or predators.

If aging is not mandatory, why is it so common in nature? There are two main theories. The first is that aging is a genetic function that is selected evolutionary since it purges individuals after their reproductive phase which means when it doesn’t affect their evolutionary fitness anymore. However this is already the first problem with this theory. Why should something so consequently be selected in so many? An argument could be that it is an advantage to make room and save resources for your offspring but that’s not really realistic in this case. The selective pressure would be different depending on the scarcity of resources but life spans are not.

The second theory is aging is just an effect of wear and unrepairable damages that accumulate. So is a body just like a car that, no mater how much you put into into repairs at some point it’s not worth it anymore? Obviously this is a factor there are measurable accumulations especially in genomic damages and epigenetic deregulation.

What speaks clearly against pure random damage leading to biological aging, is that it has a clearly distinguishable phenotype. We all are able to tell if a person is old. This seems like there are biological integrators and common pathways activated by different kinds of damage. There are two kinds of those pathways. Those that repair or prevent the damage like stress responses. And those that in case the damage becomes too much and bears the risk of turning the cell into a cancer cell. Those can be programmed cell death in extreme cases but also senescence. Senescence means that cells permanently stop deviding behave differently but stay alive.

This is responsible for many of the observable phenotypes of old age. The more senescent cells an organ contains the older the whole organ behaves.

Back to the main question: Why do we age? The answer is, that we don’t know yet. It seems however that it is a mix of wear and tear that is integrated by molecular responses to the damage and a deregulation that is due to the loss of selective pressure after the end of the reproductive phase.

If you want a deeper dive into the topic from a scientific perspective a good starting point could be the review article by Shufei Song and F. Brad Johnson (2018).

In case you’re not sure how to get scientific publications without paying the horrendous sums many publishers charge for them, stay tuned there will be an article on this topic soon.

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