Sizes in Biology

Dear colleagues,

An excellent tool is available to get a feeling about molecular sizes in Biology.

The tool is available here and was developped by "Learn Genetics" program from the University of Utah.

You can have a short movie on it below, but please check the original one from here!!!








You can also have a deep immersion in the issue here:

Green Fluorescent Protein or GFP

Green lights in the dark



When someone first shows up in our lab, the prime goal I set up for him or her is to make "green cells" - I mean to introduce a Green Fluorescent Protein into a mammalian cell culture. In order to be able to perform this one has to know some basic molecular biology. One has to know what a cell is, what the difference is between a prokaryote and an eukaryote cell; what the central dogma is namelly that the information flows from DNA to RNA and from here to proteins is, or as it has been formulated originally and still correctly, the information flows from nucleic acids towards proteins (albeit I assume we will see exceptions for this rule, too). (You can reach a very good lecture on this topic here.)  One has to know what the difference between DNA and RNA is, in most basic approach the chemical difference is minuscule (there is a deoxyribose in the backbone of the DNA and a ribose in the RNA, there are other differences but this is the most prominent), while the results are spectacular. DNA is a quite stable molecule that can be degraded by DNAses. DNases require divalent metal ions for their activity ( usually Mg, but other divalent ions can be used too), and we can remove these ions from solutions with so called chelating agents. Most commonly we use EDTA for this task.

From practical point of view, one needs to have some backgrounds in order not to be lost in a molecular biology lab as it follows:

One has to be able to use pipettes (as seen in the previous posts), to make buffers, to know about pH, know what molarity is, and have a good basic background in maths (just enough to calculate the compositions of the buffers).

But you can perform the most basic experiment of DNA isolation even in the kitchen! At the end of this experiment you will be able to even SEE the DNA!

You can extract DNA from any cell, but the easiest way is to use some germs, like wheat or bean germs, soya germs and so on... In the following video you can see the procedure. If you do not have isopropyl alcohol (I don't have at home for example) use regular ethanol or a strong spirit with at least 70% alcohol content!



Regarding RNA, the world of RNA is a transient world.  RNA is degraded by enzymes that can be found everywhere. RNAses can not be blocked by removing metal ions with EDTA. This makes the half life of RNA very short. Let us take the analology of the computer: DNA is like the information on the hard disk, one might have a software on the computer without using it- this is the information in the DNA. If one double clicks on its icon, the program starts, this corresponds to the transcription: information is transcribed from DNA to RNA, or the software is running, even if it is not yet in use, it is ready to get an input and process it into the output. The RNA is similarly translated by ribosome into proteins: these are the products that have been coded in the DNA. Or according to the computer analogy you create a document with the word processor software. The document is an entity by itself.  You can print it and have it. If you turn off your computer, the temporary files are destroyed, all unsaved files are deleted. So is with the RNA. RNA is carrying an information for a short period of time, it has a short half life, but can be regenerated from the DNA. These processes are explained in the following video:



Ok, so how do we make green cells? Green flourescent protein is encoded in the genome of the Jelly fish. The protein once identified can be introduced into other organisms if we isolate the DNA sequence that is encoding the GFP protein. So let's have a look to these wonderful organisms!

Beautiful Jelly fish



The discovery of GFP protein and their mode of action changed plenty of studies in biology. The Nobel Prize for Chemistry in 2008 was given for the identification of the GFP protein and its way of action. You can see below two videos about the topic. A detailed, in depth one or below a short overview of the topic. You choose!

Giving green light to biology



Nobel Prize for GFP



After this overview I think it is time to have an experiment. We will see how you can introduce the GFP encoding DNA into a bacteria. For this we use so called plasmids as a vector. We call vector in biology a tool that is able to carry genetic information, like a plasmid, cosmid, or a virus. A plasmid is a small circular DNA that is able to self-replicate into a bacteria and to express a protein. They are responsible for lateral gene transfer in bacteria, e.g. transfering antibiotic resistance gene from one bacteria to a different one.

In the following experiment we will see the introduction of a GFP encoding DNA into a so called Agrobacterium, a bacteria that is infecting plants.

Introducing the GFP into a bacteria



Cool, isn't it?

We can make even more complicated investigations with the help of the GFP. In the following animation it is shown the transfection process in a mammalian cell where the addressed question is if two proteins interact or not? For this they use the so called FRET or fluorescence resonance energy transfer. In order to see if the two proteins are close to each other or not, we have to use two GFP like tagged proteins with their excitation and emission wave lengths close to each other. See how it works:

Investigating protein-protein interactions with fluorescent proteins



GFP has several other applications, like tracing of migrating neurons, as seen in the following video:



Or full GFP organisms like in the following one:



If you would like to know even more about the GFP protein, please visit the best site in this topic I have ever seen, the page of Marc Zimmer, here.

I think we had even too much of GFP now, so in the next posts we will go back to plasmids...

See you!

Why Molecular Biology?

At the very end you might ask why is the life in a molecular biology lab so interesting?

We discussed about water, pipettes and we will go on with several topics, but at the very end there is a wonderful, miraculous world. Each cell in our body and each cell in any living organism works based on the same principles. Information is stored, processed and replicated in cells.

If we could have an insight into these processes we could better understand what is life. Yes, I think this is still a question! What is life? How can you explain the abundance seen on every cubic centimetre of the surface of this planet?

Instead of giving a flat answer, let us look to the best animation I have ever seen about THE INNER LIFE OF THE CELL!

Here it is:

Water in the Lab

Hi,

Before we make the first experiment we have to discuss about some trivialities that might be different in the lab than in the outside world.

For example: water. Everyone knows what water is and I don't want to recapitulate again the basics. You can have a real good overview here.

We use water for plenty of applications in the lab. Some of them are not specific to the lab world. Here are some examples:

Of course we use water for various lab specific purposes. The most important of these purposes is to prepare various solutions. In order to control as much as possible how our solutions will work we need a realy pure water. Tap water although is considered as pure drink water contains plenty of soluble components like: ions, colloids particles and so on. This water can not be used to prepare solutions. We use it to wash dishes but even after dish washing all dishes has to be rinsed with ion exchanged water. Ion exchaged water replaced distilled water in the last decades and stands for water that contains almost no ions at all. Distilation was used earlier to evaporate and ... water and by this procedure you can get rid of the soluble salts from the water. The procedure was simmilar to the destilation of alcohool in distileries like this. The ion exchange resins are able to bind the ions from the water and produce a water that has the same qualities as distilled water has.

But how do you know if a water is pure?

It was told that you shoud use your senses: like smell it, view it, taste it. A clean water should be clear, tasteless and should not smell. But this is not enough. The easiest way to measure the presence of ions in water is by measuring its electrical conductivity. Soluble ions in the water will allow electricity to pass through the water. A really pure water is having very low conductivity.

In our lab we have a special tap for central ion exchanged water:

So don't worget, after washing lab dishes, please rinse everything at least twice with the ion exchanged water from this tap!

Can we use this water for solutions?

In some cases we could. Nevertheless due to the fact that we process sensitive biological samples like DNA and proteins we do not use this water for solutions in a molecular biology lab!

In order to prepare water for solutions we use so called "MilliQ" water. We introduce the ion exchanged water into an apparatus which is filtrating it through a replaceble cartridge. This filtrated water is free from colloids, proteins, ions and is suitable to be used in regular molecular biology solutions. Of course not for all applications! We will discuss this later. Here is the instrument that is producing the "Milli Q" water:

You will find the water for solutions right in in a plastic carboy (also called demijohn) like this:

You can use this water for preparing buffers for gel electrophoresis, western blot and so on.

By sterilizing it, you can make sterile solutions for cell culture applications. Nevertheless I would stronglly recommend that you should filtrate these solutions through a 0.2 micrometer filter. Majority of infecting agents (from bacterial origin) are larger than 0.2 micrometers so a sterilizes and/or filtered solution should be OK for cell culture applications.

There are some special applications that need special waters.

Two of them are RNA applications and cell culture applications for immune studies.

1. RNA applications.

While DNA can be protected quite easilly by adding EDTA as a chelating agent to the solutions (by this you get rid of the soluble Mg and other ions and you block the activity of DN-ases) RNA can not be protected like this. RN-ases are everywhere and are destroing the free RNA. That means that we have to use a special water that has no active RN-ases. Earlier we used so called DEPC treated water. Now we we use so called "Nuclease free water". Earlier we were buying it in small 25ml bottles like this:

Now we buy it in larger quantities and alliquot it. We use this water as NFW (Nuclease Free Water):

As a rule: USE ALLWAYS YOUR OWN NFW!!! Mark it with your name, and put a date when you oppened the tube.

2. The second type applications when we need an even purer water are the immunologic studies. In these cases we need a water thet is free of LPS (bacterial lipopolysaccharides, or endotoxins). The water we use for these applications is called "Embryo water" although we do not use it for embryological manipulations, it is LPS free. It is very important to alloquote it only in endotoxin free tubes, like cell freezing sterile vials.

Here is our LPS free water:

So these are the water types in our lab. We will discuss about the price of our water types later!