(Words that are highlighted can be found in the biotechnology definitions page by clicking in the word or following the link at the page end.)

Biotechnology is basically the use of biological cells or systems to make particular compounds. Most commonly it is micro-organisms doing the manufacturing because they are very versatile chemically, grow rapidly and to large numbers and produce many compounds that we cannot synthesize any other way.The use of micro-organisms in biotechnology is not new. Perhaps the oldest examples are making bread, yoghurt, cheese, or beer . The production of antibiotics such as penicillin and streptomycin rely on microbial biotechnology. However recently we have entered a new revolution in which new opportunities have developed. For example we can now use bacteria to make human proteins such as insulin instead of extracting it from the pancreas of slaughtered pigs. Human growth hormone to treat dwarfism is made in a test tube not extracted from brains in incredibly small amounts. Why this change? What is the basis for the new biotechnology industry?

The key to the development of biotechnology has been our understanding of DNA structure, its relationship to the genetic code and, most recently, the development of recombinant DNA technology allowing specific genes to be isolated, altered and transferred. Although the basic principles of heredity were established last century, largely by Gregor Mendel, it was not until the 1960s and 1970s that the role of chromosomes and DNA, and the genetic code as the basis for heredity were established. We know now that genes are composed of DNA. However the structure of the DNA molecule was elucidated by Crick and Watson in 1953. This suddenly allowed the understanding of how DNA could replicate and encode all the incredible amount of information associated with even the simplest unicellular organisms.
 
 

DNA Structure

DNA consists of 2 complementary strands of nucleotides consisting of compounds called bases attached to a sugar (deoxyribose). The sugar/base units are linked together by phosphates. There are 4 different bases. : ADENINE, THYMINE, GUANINE and CYTOSINE

 
 
 
 

Adenine and guanine are purines and cytosine and thymine are pyrimidines. The complementarity or blueprint is provided because specific pairs of a purine and pyrimidine are linked together. Only the combination of a purine and pyrimidine will fit together to link the two single stranded molecules into a double strand. Adenine (A) and thymine (T) guanine (G) and cytosine (C).
 
 
 
 

DNA Replication.

During cell division a copy of the total DNA molecule must be generated for each daughter cell. This process is called REPLICATION. During replication the two strands of the helix separate in a particular area and a new complementary strand is synthesized. Thus each new DNA molecule is semi-conserved (one strand is parental and one strand new). The enzymes responsible for this process are DNA polymerases. The synthesis of an exactly complementary sequence is achieved because only one base can be fitted adjacent to the parental strand base. Sometimes mistakes will be made during this process - and an incorrect base pair gets fitted or a base omitted - mutation.

Transcription

The DNA structure encodes for proteins. Proteins are manufactured from amino-acids. All the different proteins in our bodies are constructed from about 20 different amino-acids put together in an almost infinite variety of sequences. These proteins may be structural (e.g. muscle) or involved in controlling various systems and processes (e.g. enzymes, hormones, immunoglobulins). In all cases the steps involved in their synthesis is identical. However the mechanisms involved in controlling their production will be very different and often complex. Not all the DNA sequence in a particular gene will be responsible for making the particular protein. There are what are called non-coding regions or introns , short sequences which do not encode for a part of the protein. The coding parts of the gene are called exons. The first stage in protein synthesis is called transcription. Transcription is the manufacture of a single strand of ribonucleic acid (RNA) from one of the parent DNA strands encoding the message in the sequences to make a particular protein. During transcription the DNA strands separate and an enzyme called RNA polymerase produce a single complementary strand of nucleic acid. This is called messenger RNA (mRNA). mRNA has a very similar structure to the single unwound strand of the DNA helix. However there are two important chemical differences. In RNA the sugar is ribose rather than deoxyribose and the base thymine of DNA is replaced by uracil. After a messenger RNA copy is produced and the DNA helix then joins back together. All this process occurs in the cell nucleus. Once transcription is complete the mRNA moves out of the nucleus into the cytoplasm where protein synthesis occurs.

Translation or Protein Synthesis

The mRNA strand has a sequence of bases complementing the structure of the DNA strand from which it was synthesized. Each triplet of bases, called a codon, defines a specific amino acid of the 20 or so different amino acids which are the building blocks of every protein. For example a series of three bases, uracil, cytosine and adenine or UCA is the code for the amino-acid serine. AAA is the code for lysine , GCU for alanine and so on. Thus the mRNA molecule forms the template for protein synthesis.

Click here to see diagram of transcription, translation and protein synthesis

Making Recombinant Molecules

A recombinant DNA molecule is one in which DNA from different sources have been combined The keys to manipulating DNA to form recombinant DNA molecules have been bacteria. Bacteria contain small pieces of double stranded DNA in closed loops called plasmids. These pieces of DNA carry few genes. They commonly carry genes for antibiotic resistance and are easily transmitted between cells and transfer to the recipient cell the properties encoded in the genes on the plasmid. These plasmids have become the delivery system for moving genes about in their manipulated form.

For a diagram illustrating the  construction of a recomibnat molecule click here.

Bacteria also produce enzymes called restriction endonucleases. These enzymes recognize particular base sequences of a DNA molecule and cut the DNA in a very precise and repeatable way. Many of these endonuclease will cut the double stranded DNA in such a way as to leave single stranded sequence of a few bases. These are called sticky ends. The critical process in all biotechnological manipulations is the construction of recombinant molecules using plasmids and restriction endonucleases. Lets assume that we want to make human insulin in large quantities in bacteria. In the first stage the insulin gene has to be identified and then isolated from some human DNA. This will probably be done by actually isolating the mRNA from cells actively making insulin in the pancreas and then using an enzyme called reverse transcriptase to make a DNA copy of the RNA. This is exactly the reverse of the natural process of transcription - hence the name. This piece of copied DNA called cDNA, or copy DNA, can then be inserted into a bacterial plasmid. Both the plasmid and the cDNA are cut with the same restriction endonuclease so they have complementary sticky ends which will allow them to stick together.

The plasmid contains the insulin gene and also genes encoding antibiotic resistance, perhaps ampicillin. It is now a recombinant molecule which will be introduced into a suitable bacterium, usually E.coli with no antibiotic resistance. The bacteria are then grown up and then selected using ampicillin. The bacteria able to grow must contain the plasmid because they are resistant to ampicillin. They will hopefully also make insulin. The bacteria can then be grown up in large vats or containers in liquid medium and as they grow they will produce insulin which can be extracted from the medium.

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