How Did Recombinant DNA Start? Cutting and Pasting of DNA Molecules in the Laboratory

A series of critical scientific discoveries were required before the manipulation and propagation of engineered DNA molecules could be undertaken.

The DNA molecule was discovered in the mid-1800s. It was not proven to be critical to the mechanisms of inheritance until the 1940s. Since the 1950s, and the seminal work of Watson and Crick to characterize the chemical structure of DNA and how it could be copied, the understanding of the biochemistry of DNA and the ability to manipulate it in the laboratory has grown exponentially. Breakthroughs studying DNA now come fast and furious, but several key discoveries are largely responsible for the development of modern recombinant DNA technology.

The Genetic Code

Knowing that DNA is the molecule of heredity is one thing. Knowing how it maintains and transmits crucial genetic information is something else entirely. In the early 1960s, Marshall Nirenberg and colleagues working at the US National Institutes of Health cracked the genetic code. Using synthetic RNA molecules and radioactively labeled amino acids they proved that DNA used a three letter code (three nucleotide bases made a “codon”) to specify individual amino acids, the codons in RNA molecules were contiguous, they did not overlap, and there were punctuation marks, places specifying “start” and “stop”.

Episomes and Plasmid DNA

In the 1950s and 1960s, numerous scientists were studying how DNA was handled in bacteria, viruses and yeast. From their combined work came the knowledge that bacteria could carry small, circular molecules of DNA that were not integrated as part of their chromosomal DNA. Most important of all, these “plasmids” or “episomes” could be isolated and more importantly they could be transferred back into other bacteria.

Restriction Endonucleases

One of the key factors in recombinant DNA technology is the ability to “cut” DNA molecules and to be able to “paste” pieces together, often in a new order. Werner Arber, a Swiss microbiologist, was the first to recognize that there were enzymes that would cut DNA molecules in specific ways. Shortly after this work was published studying the bacterium E. coli, Hamilton Smith and colleagues identified a “restriction endonuclease” from another bacterium, Haemophilusinfluenza, and then showed that it cut at a very specific sequence of DNA bases. Today there are hundreds of restriction enzymes known to cut DNA at specific occurrences of base sequences.

Recombinant DNA Molecules

In 1972, scientists Herbert Boyer and Stanley Cohen were in Hawaii attending a scientific meeting about plasmids when they met to discuss the work that they were each pursuing. What ultimately came from this meeting was the birth of recombinant DNA cloning, the start of the biotechnology industry and a new era in molecular biology. Collaborating on the use of newly identified restriction enzymes and DNA manipulation techniques, they published seminal papers showing that DNA from one source could be cut with restriction enzymes and then placed into the midst of cut DNA from another source, and these could be placed into bacteria and grow stably with the “recombined” DNA maintained in the newly arranged form.

Herbert Boyer went on to found the biotech company Genentech and the research world has never been the same. Recombinant DNA methods are now used to make things from insulin for diabetics to proteins that can make individual cells, or even whole organisms, glow green, blue or red under just the right light. Remarkable.

To read more about the cracking of the genetic code by Nirenberg and colleagues visit the history page at the US National Institutes of Health.

Antibiotic Susceptibility Tests – Types and Ways: Antibiotic Evaluation, Antimicrobial Effectiveness In Vitro In Vivo

Proper antibiotic susceptibility testing of microbial pathogens isolated from patients is critical. The use of an ineffective antibiotic could lead to a patient’s death

Today, if someone gets a bacterial infectious disease the first thought is “what antibiotic will help or work?” The doctor and the patient both desire the best and most effective antibiotic. This is what makes testing and analysis of antibiotics very interesting.

In the 1950’s and 60’s there were sufficient antibiotics on the medical scene to require a closer look at standardized testing of antibiotics. A better way was sought to accurately and precisely forecast which antibiotic would work and which would not.

Drs. Kirby, Bauer, and Sherris established a defined scientific approach to antibiotic testing (KBS technique) . This greatly improved the accuracy and results of testing. There are several important ways to determine antibiotic susceptibility.

Solid, Liquid and Animal Tests of Antibiotics

  • When chemicals and antibiotics were first tested they were added to nutrient agar petri dishes (circular glass, or plastic, with larger top glass cover overlapping a bottom reservoir) with bacteria streaked over the surface. The next day a zone of inhibition was looked for as shown in figures below (click to enlarge these figures).
  • Bacteria can also be tested in test tubes with nutrient broth. Sequential dilutions of the chemical or antibiotic are done first, followed by addition of a measured bacterial inoculum to each tube. After overnight incubation the tubes are analyzed: clear tubes indicate inhibition and cloudy tubes indicate bacteria not inhibited by that concentration or titer of antibiotic. In this way scientists can correlate later how much antibiotic yields what zone size on agar.
  • The zone sizes produced on plates by dropped antibiotic disks are plotted versus the amount in the disk. Simply, this is a regression analysis graph. It is possible to establish MIC (minimum inhibitory concentration) values in broth, blood or serum in this way. Typically, the more resistant a microbe is the smaller the zone. The more susceptible, the bigger the zone.
  • Finally, if an antibiotic looks promising, it can be tested in mice or rabbits for toxicity (harmful) and effectiveness.

Measurement of the amount of antibiotic injected, or ingested, is the dose. The antibiotic titer is the MIC = minimum inhibitory concentration ( an actual value attainable in blood or serum). MIC is measured in micrograms or units. MIC can be evaluated in blood and tissue fluids . The higher the MIC the more resistant the bacterium is to that antibiotic.

Standardized Antibiotic Testing

Antibiotic testing should always be done with a pure culture of the organism.

The KBS technique eliminates the variables that affect the zone size and could cause false positives or false negatives.

The agar used is Mueller-Hinton. The depth and pH of the agar,inoculum size and incubation temperature are standardized. The click-on photos below show several things:

  • different antibiotics have characteristic zone sizes for MIC
  • the same antibiotic at different concentrations will give different zone diameters
  • resistant bacteria cause zone size decreases. No zone = complete resistance
  • regression analyses enable microbiologists to determine MIC vs disk zones
  • populations of the same species vary from susceptible (S) to intermediate (I) to resistant R)

Recently, the valuable E-test (see photo) allows determination of MIC of a microbe with one disk that forms an antibiotic gradient. This permits rapid, clear medical evaluations of antibiotic effectiveness.

Review of the Primer of Conservation Biology: A Teaching Tool on the Topic of Conservation Biology

Learning about conservation biology is the first step to making a difference, and through the text by Richard B. Primack that becomes possible.

Conservation biology is the field that seeks to study and protect the living world and its biological diversity. So says Richard B. Primack at in the prefix of his book called A Primer of Conservation Biology. His intent with that statement is to provide a specific definition of what conservation biology is, and to lay the beginning framework of what the rest of his book is about.

What is Conservation Biology?

Conservation Biology is still a relatively new field, because there hasn’t always been a movement to save the Earth. Resources are burnt thorough quite quickly, and in doing so the environment is hurt in more ways than one. No matter what side of the “environmental argument” someone’s on, there is debate that the planet is being harmed more every day. What Primack hopes to do with this book, is to show what is happening, and what people interested in the field can do about it.

Thoughts on Conservation Biology

The first part of the book describes conservation, and why it is needed. Resource management is a huge step in the right direction and the methods to do it are explained with depth. This is a great section about exactly what is being done wrong, and where the mistakes can be fixed if subtle changes are made in the way that people do things. The text relates about extinctions that are taking place all the time, and the rates at which those extinctions are occurring.

From there he goes into discussions about the threats to biological diversity that are beginning to happen as more and more species are threatened. Those aren’t just limited to the larger animals that are in the news, but plants, bugs, and even microscopic creatures that depend on other species to keep living. He also gives examples of why these extinctions are happening.

Putting Conservation Biology into Practice

Conservation at the population and species level, and conserving biological communities are what Primack dives into as the book progresses. He shows how designing networks of protected areas, and managing them, along with ecological restoration of those areas that some of these problems can be slowed down. Conservation isn’t something that can be implemented right away, and it’s key to take the steps and put the procedures in place so that it can become something that everyone focuses on more.

Through the roles of agencies (both public and private) and through Government programs conservation is becoming something more center-stage to the public. The key here is to learn as much as possible about the field to get involved in helping conservation, and Primack’s book is a good stepping off point.

Primack explains the terms well, and the theories and facts of everything he talks about are phrased so even the most novice reader can understand. The entire book does a great job of telling the reader the basics of conservation, what has been implemented so far, and what still needs to be done for it to all work out. Conservation Biology is an important field that is slowly becoming more popular as people realize just how badly the environment is being treated.

Final Recommendation on the Primer of Conservation Biology

For those interested in anything to do with conservation, this is a great tool to provide knowledge that would be necessary to be active. The book comes in at 320 pages, is paper back, and can be found online or in most bookstores. It is highly recommend it to anyone wanting to read more about conservation biology.

The Mechanics of the Neck and Skull: This Structure Protects the most Vital Organs in the Body

The bones of the skull form the structure of the face and are arranged to safeguard the fragile tissues of the brain and spinal cord.

Apart from acting as a protective casing for the brain, the head has many more important jobs. The bones and muscles of the skull, face and neck help movements such as:

Turning, Nodding, Chewing, Swallowing, Looking, Listening, Breathing, Talking and Subtle facial expressions

The bones of the skull are separated into two main groups. There are eight bones that form the cranial vault which protect and support the brain. The cranium is held together by bands of fibrous tissue called sutures. 14 other bones are responsible for structuring the skeleton of the jaw, cheeks, eyes, ears and nose.

Cavities that are full of air, known as the sinuses, and a honeycomb of air-filled pockets in the mastoid process, help to lighten the weight of the skull. Little holes in the cranium allow blood vessels and nerves to move through to structures on the skulls surface.

The Structure of the Nasal Cavity

The nasal cavity is separated by a bone and cartilage septum. Bony projections called conchae disrupt the flow of incoming air, meaning it will bounce around the cavity, dropping dust and germs in the mucus lining. Draining into the cavity is a number of air-filled chambers lined with mucus secreting membranes.

There are three tiny bones that conduct sound waves between the ear drum and the inner ear. These are known as the ossicles, with the smallest of these, the malleus, measuring just 8mm in length.

The Mechanism of the Jaw Bones

The jaw bones consist of a large lower bone, the mandible and two upper bones called the maxillae. How the jaw bones fit together is known as occlusion. The temporomandibular joints allow the lower jaw bone to connect to the skull meaning opening, closing and sideways movements can be made when talking or chewing. These are the only moveable joints within the skull structure.

The teeth are held in a fixed position in the jawbone with fibrous sheets of connective tissue. Each tooth is covered in a hard outer shell of enamel above the gum, with a bone like cementum forming the outer layer of the tooth below the gum.

The spinal cord passes out of the head through a hole at the bottom of the skull and down the vertebrae in the neck. The throat is made up from the trachea and larynx and is supported by a group of small bones and cartilage rings in the front of the neck.

The Bones that help to form the Neck

The neck contains seven cervical vertebrae, including two specialised vertebrae, the atlas and the axis, at the top of the spine. The atlas helps to support the weight of the head and takes it name from the mythological giant Atlas, who was thought to carry the weight of the world on his shoulders. This vertebra allows the movement of nodding of the head. The axis vertebra forms a pivot joint around which the skull can rotate from side to side.