Penicillin

The discovery of antibiotics is one of the most significant medical accomplishments of the last century. Their impact has been profound. Along with improvements in sanitation and vaccinations, the use of antibiotics remains at the forefront of our largely successful efforts to control infectious diseases. As with PCR, we owe the availability of antibiotics to evolution. They are not invented, they are produced naturally by a variety of different organisms.

The modern era of control of bacterial infections began in 1927 with the discovery by Alexander Fleming that extracts from the mold Penicillium notatum lysed bacterial cells. He was not able to isolate the active compound but gave it the name Penicillin after the mold’s genus. The active compound was eventually isolated and developed into a therapy in the early 1940’s by a groups led by Chan and Florey. For their efforts, they shared the 1945 nobel prize for physiology and medicine with Fleming. The receipt of the Nobel Prize just a few short years after the introduction of penicillin as a drug speaks to the impact this medication had.

Many new antibiotics have been introduced since the 1940's but Penicillin and penicillin like antibiotics remain in wide use, comprising almost 20% of all antibiotics manufactured.

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Nature's contribution to modern science

The polymerase chain reaction or PCR is central to much of the molecular biology research performed today. The technique was used for the human genome project, is used as the definitive test to identify many pathogens including H1N1 and is the basis for our understanding of the tree of life. Hundreds of thousands, if not millions of PCR’s are performed every day in labs all over the world. The enzyme used for PCR is called Taq Polymerase¹. It is at the heart of the PCR reaction and it was not engineered by man. Rather, it evolved billions of years ago. It is a beautiful example of natures power to innovate.

Polymerases are a class of enzyme that catalyze the synthesis of a chain, or polymer, from component parts. Taq polymerase is a DNA polymerase, catalyzing the syntheses of DNA from nucleic acids. All living things have to have at least one DNA polymerase in order to synthesize the DNA required for growth and reproduction.

The exponential amplification characteristic of PCR stems from ability to repeat the reaction steps over an over again by cycling the reaction through a series of temperatures. These temperature steps include one at near boiling. This high temperature separates the double strands of DNA allowing each strand to be used as a template to synthesis more DNA.

Most enzymes (including most DNA polymerases) are not heat stable. They denature when heated and lose their activity, even after they are cooled back down. Taq polymerase isolated from the thermophilic bacteria Thermus aquaticus retains it activity after being heated to near boiling (95 °C). It is not active at 95 °C, but renatures and regains its activity upon being cooled. The ability of Taq polymerase it to remain active after repeatedly being heated allows the cycling of the PCR to create billions of copies of a target sequence from a single template using reagents added at the begining of the reaction.


¹This was the first theromostable polymerase to be widely used. In the more than 25 years since its introduction, other heat stable polymerases have been introduced.

*last edited 11 May 2010 - minor edits and links added

PCR

It is an exciting time to be following progress in the field of microbial ecology. While the awareness of the abundance of microscopic organisms is not new, the development of new methods to observe the microscopic world continue to deepen our understanding. Some of these methods have been described briefly on this blog. See the entry on 454 sequencing for example.

One of the most important techniques for modern microbial ecology and biology as a whole is the polymerase chain reaction or PCR. I suspect most of my modest readership is familiar with the technique, if any of you are not, it is something that you ought to take the time to learn about. A google search of the term PCR reveals a large number of pages devoted to explaining the technique. Many people have also developed diagrams and animations to aid in the understanding. The problem is, most of the good animations do not stand on their own. To make use of them, some background knowledge is needed.

The best animation I have seen on the web is this one. Go ahead and look at both the amplification animation and the interactive graph showing the number of copies of the target molecule present after each cycle.

Here are some things to keep in mind:

1. DNA is a double stranded molecule and the two strands are held together by hydrogen bonds. Each individual bond is weak, the strength of the bonding between the two strands arises from the sheer number of individual bonds present. Key point for PCR: The bonding that holds the double strand together is easily disrupted by heat. Thus the 95 deg C steps
2. DNA is made up of 4 nucleotides: adenine, thymine, cytosine and guanine attached to each other along a sugar-phosphate backbone. The hydrogen bonding between the two strands of the double stranded molecule are between these nucleotides. The pairing of the nucleotides is specific. Adenine always binds with thymine and guanine with cytosine. Key point for PCR: knowing the sequence of one of the strands of the double stranded DNA makes it possible to deduce the sequence of the opposite strand.
3. DNA polymerase is the enzyme required for PCR. The enzyme is capable of synthesizing double stranded DNA from single stranded DNA using the single strand as a template. The activity of this enzyme is specific in several ways. Most importantly for PCR:
• The nucleotide bases strung along the sugar-phosphate backbone of each DNA strand has directionality.^a and each of the two strands in the double stranded molecule are oriented in the opposite direction. DNA polymerase can elongate in only one direction. Key point for PCR: the DNA polymerase must elongate each of the two strands from opposite ends.^b
• DNA polymerase can not elongate single stranded DNA. A short fragment of double stranded DNA is needed. Key point for PCR: Small lengths of double stranded DNA need to be created flanking the region targeted for amplification (these are the primers).
4. The exponential nature of PCR amplification depends on multiple cycles of amplification involving both strands of the double stranded molecule. Key point for PCR: Two primers are needed.
   

The ingredients needed for a PCR reaction are:

• DNA polymerase - the enzyme.
• 2 primers - Small fragments of single stranded DNA. These are used to produce short regions of double stranded DNA flanking the sequence targeted for amplification.
  
• Free nucleotides - These must be in the form of nucleotide triphosphates. In this form, they provide the source of the bases needed to build new strands of DNA and the energy required to drive the reaction.
• Template - some DNA containing the region that is to be targeted.

When I teach people the basics of PCR, a question I use to assess comprehension is: How many cycles of amplification are needed to produce the first copies of the target fragment that are the correct length and why are are none produced prior to this cycle? If you can answer that question, you understand much of the basics of PCR.

There is much more to say about PCR but this post is already long enough so go enjoy the animation.

^aFor a better understanding of the structure of the DNA molecule itself see DNA is a polynucleotide by Larry Moran at Sandwalk.
^bSee here and here for an interesting exception to this rule on directionality.

Image from Flickr

Nitrogen fixation

Industrial agriculture is highly dependent on a ready supply of labile nitrogen fertilizer for high yields. Roughly half of all the nitrogen used in agriculture is in the form of ammonia (NH₃) synthesized using the Haber-Bosch process. In this process, atmospheric nitrogen in the form of N₂ gas is mixed with hydrogen gas at high temperature (500 ^oC) and pressure (200 atm) in the presence of an iron catalyst. That's 500 ^oC and 200 atm. Very hot and very high pressure.

Bacteria (and archaea) are also capable of converting nitrogen gas to ammonium (often referred to as 'fixing nitrogen') but they do it at normal temperatures and under 1 atmosphere of pressure. In these organisms the process is also energy intensive but the energy is supplied in the form of ATP and the catalyst is an enzyme complex containing 2 proteins, dinitrogenase and dinitrigenase reductase. This capacity is found in both bacteria and archaea in diverse environments including the root nodules of so called nitrogen fixing plants such as clover and soy. Bacteria, not the plants, fix the nitrogen.

There are many blog worthy aspects of microbial nitrogen fixation. I have created a label for this topic and intend to explore some of them here in the future.