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.
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.
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.
It’s been about two weeks since I started actively using this blog and as you’ve probably seen I’ve been busy. While I started this blog, I also started to think about how to get people to read it. The most obvious answer is social media. Social media channels are made to engage people and show them what you find interesting. For a long time I’ve only been using Facebook for private and LinkedIn for work purposes. I never really used Twitter because I always thought it’s mostly a platform where politics and media exchange views and act like they are the public.
Than I had a seminar about science communication with Susanne Geu. She told me Twitter is an excellent platform for science communication so when I started the blog, I also started to become active on twitter.
After about two weeks of active work on this blog and different social media channels I made the following discoveries
When it’s about clicks on your blog forget Twitter. You can very quickly find a great bubble of people working in a similar area and engage them quickly (from 1 to 70 followers in one week) but people on Twitter like it short. From thounsands of impressions you get a few hundred interactions and from those only a handful are clicks on your links. However it is still a great tool to get in touch and to communicate punchlines.
LinkedIn works about as good as I expected. Since I have a lot of contacts there I get a lot of quite some likes and quite some clicks but no active discussion or interaction.
I originally wanted to keep Facebook private but I still posted my Blog so friends could see what I’m doing and that obviously brought some encouragement. To tell friends, family and people you know what you’re doing you can also use the WhatsApp status.
What works really well to get clicks but also really good discussions is groups in Facebook. Find some good science groups and post and discuss there. This is not too easy because most groups in Facebook are either full of spam or completely inactive but there are some.
I’ll write an update on my experience with social media in a few months maybe then my impression has changed but so far I’d say all these channels have merits and a different target group.
What I did’t try yet because I do not really thinks that’s my audience is Instagram and TikTok. But maybe I’m wrong there so if you have a different opinion on these networks please let me know.
If you want to see what I do on these platforms please follow me on LinkedIn or Twitter!
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.
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.
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:
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.
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 firsthand scientific information is hard. First most articles use scientific slang that is hard to understand if you are not specialized in this field. However it is often worth it since you get rid of the filter of normal news journalists who often do not really understand the papers themselves and sometimes really do not get the right point. If you regularly read popular science pages you might have noticed that the authors of those articles are often a bit overly enthusiastic about some research results. They overstate because click baiting is a way of earning money with your news site.
Since that is not the case with scientific papers (they do not normally earn money for their authors) their tone is a lot more honest.
Now if you are looking for scientific papers you can use Google but that is not very specific. One of the most standard search pages for scientific papers is pubmed and it does this quite well. But after you found an interesting article here it often puts you in front of the next barrier to enlightenment, namely a pay wall.
To avoid these your first step is back to Google. However not to the normal search but to Google Scholar. The main difference to Pubmed is that on the right side of the search results Google shows you other pages with the same article and especially those where they are available for free.
This already helps a lot but there are still some articles that Google Scholar won’t offer you for free. The next tool to also find most of them is Sci-Hub. This is a tool that works in a bit of a grey zone. It collects articles from authors who, if you contact them directly, normally gladly share their manuscripts with you for free.
I think it is totally legitimate to offer free access to research that in the overwhelming majority has been paid for by public money.
Now you should have access to almost all scientific papers you need. If you still find an article that is not available for free with these tools, you should try to contact the authors (contact details should be directly below the title even in the preview). Most of them will be happy about the interest in their research.
I’ll later write a post about how to organise and store all your newfound scientific knowledge.