Biotechnology: Principles and Processes

Biotechnology uses living organisms (or their parts) to make useful products. Its heart is recombinant DNA (rDNA) technology — cutting a desired gene from one organism and joining it into the DNA of another. This needs a specific set of tools, and the most important are the enzymes.

Restriction enzymes (restriction endonucleases) are the 'molecular scissors'. Each recognises a specific short sequence (recognition site), usually a palindrome (reads the same 5'→3' on both strands), and cuts the DNA there. Crucially, many of them cut the two strands a little apart, leaving short single-stranded overhangs called 'sticky ends'. Because a vector and a foreign DNA cut by the same restriction enzyme have complementary sticky ends, they can base-pair and be joined together. A famous example is EcoRI (from E. coli); restriction enzymes are named after the bacteria they come from. The sticky ends are then sealed by DNA ligase, the 'molecular glue' that joins the two DNA fragments.

The recombinant DNA must be carried into a host cell by a cloning vector — a DNA molecule that can self-replicate in the host and carry the foreign DNA. The commonest vectors are plasmids (small, circular, extra-chromosomal DNA of bacteria) and bacteriophages; pBR322 is a classic plasmid vector. A good vector must have three features: an origin of replication (ori) so it multiplies in the host; a selectable marker (such as antibiotic-resistance genes) to pick out the cells that took up the vector; and recognition (cloning) sites where restriction enzymes can cut to insert the gene.

Finally, a host (competent) cell — usually the bacterium Escherichia coli — receives and multiplies the recombinant DNA. (Other useful tools include reverse transcriptase, which makes DNA from mRNA.) For NEET, fix the four tool groups: restriction enzymes (cut at palindromes → sticky ends, e.g. EcoRI), DNA ligase (joins), cloning vectors (carry; ori + selectable marker + cloning sites; pBR322) and the host (E. coli).

Even with the gene joined into a vector, the recombinant DNA must get inside a host cell to be cloned. But cells — with their hydrophobic membranes — do not normally take up large, hydrophilic DNA molecules. So the host must first be made 'competent' to receive the DNA.

A standard chemical method makes bacteria competent using a divalent cation, usually calcium (CaCl₂): treating the cells with calcium increases the efficiency with which DNA enters through pores in the cell wall. The recombinant DNA is then added, and the cells are given a brief heat shock (about 42 °C) followed by cooling on ice; this heat-shock treatment helps the DNA pass into the cell. A bacterium that takes up the recombinant DNA is said to be transformed, and the process is transformation.

Several other methods physically deliver DNA. Micro-injection directly injects recombinant DNA into the nucleus of an animal cell. The gene gun (biolistics) bombards plant cells with high-velocity micro-particles of gold or tungsten coated with DNA — a popular method for plants. For plants, a 'disarmed' (non-pathogenic) Agrobacterium tumefaciens can also be used as a natural vector to deliver genes.

Once the recombinant DNA is inside, the host cell replicates and expresses it; growing many such cells gives many copies of the gene (and its product). To select the transformed cells, the selectable markers on the vector are used — for example, only cells carrying the vector survive on a medium with the relevant antibiotic. For NEET, fix the meaning of 'competent' cells, the CaCl₂ + heat-shock method (and the term transformation), and the alternative delivery methods (micro-injection, gene gun/biolistics, disarmed Agrobacterium).

Making a recombinant product follows an ordered sequence of processes. The first is isolation of the genetic material (DNA). Because DNA is enclosed within membranes, the cells are first broken open and treated with enzymes — lysozyme (for bacterial cell walls), cellulase (plant cells) or chitinase (fungal cells) — and proteins and RNA are removed with protease and ribonuclease. The purified DNA is then precipitated out by adding chilled ethanol, where it appears as fine, collectible threads.

Next comes cutting (digestion) with restriction enzymes. Both the source DNA (carrying the gene of interest) and the vector are cut with the same restriction enzyme so that they have complementary sticky ends. The progress of cutting can be checked by gel electrophoresis, in which DNA fragments are separated by size in an agarose gel under an electric field — smaller fragments move farther toward the positive electrode (DNA is negatively charged), and the bands are seen under UV after staining with ethidium bromide. This step also lets the desired fragment be cut out and recovered (elution).

The cut gene and vector are then joined by DNA ligase to form the recombinant DNA. To get usable amounts of the gene, it is often amplified first by the Polymerase Chain Reaction (PCR), an in-vitro method that makes millions of copies of a target DNA segment. Each PCR cycle has three steps: denaturation (heating to ~94 °C to separate the two DNA strands), annealing (cooling so that two short primers bind to the ends of the target region) and extension (a DNA polymerase synthesises the complementary strands).

The key enzyme of PCR is the heat-stable Taq polymerase (from the thermophilic bacterium Thermus aquaticus), which survives the high denaturation temperature, so the same enzyme works through every cycle. Repeating the cycle about 30 times amplifies the DNA roughly a billion-fold. For NEET, fix the isolation enzymes (lysozyme/cellulase/chitinase; precipitation by chilled ethanol), gel electrophoresis (size separation, ethidium bromide/UV), and PCR's three steps with heat-stable Taq polymerase and primers.

After the recombinant DNA is made (and amplified), it is inserted into the host cell, which is made competent as described earlier. Once inside, the recombinant DNA multiplies and expresses itself, and the host cells are grown so that many copies — and the protein product — are obtained. The transformed cells are selected using the vector's selectable markers.

To obtain the product on a large scale, the cells are not grown in small flasks but in bioreactors (fermentors) — large vessels (often 100–1000 litres or more) in which cultures are grown under controlled, optimal conditions of temperature, pH, oxygen (aeration), substrate (nutrient) supply, agitation and removal of products/wastes. The most common type is the stirred-tank bioreactor, which has a stirrer (to mix and aerate), an oxygen-delivery system, a foam-control system, temperature- and pH-control systems, and sampling ports. Bioreactors allow the production of useful proteins/enzymes in commercial quantities.

The final stage is downstream processing — everything done after the biosynthesis (fermentation) is over to turn the crude culture into a finished, marketable product. It includes separation and purification of the product from the cells and medium, followed by formulation with suitable preservatives. For a drug, this is followed by clinical trials and rigorous quality control testing.

Downstream processing and quality control are product-specific (they differ for each product) and form a major, often costly, part of biotechnology manufacturing. So the complete cycle of rDNA technology is: isolate DNA → cut with restriction enzymes → amplify (PCR) → ligate into a vector → transform a host → grow in a bioreactor → downstream processing to obtain the final product. For NEET, fix the role of the bioreactor (large-scale, controlled growth; stirred-tank parts) and downstream processing (post-fermentation separation, purification, formulation, QC; product-specific).