Biotechnology & Genetic Engineering

Biotechnology is the use of living organisms and systems - or parts of them - to make products that benefit people and the biosphere. Its four broad areas are health care (medicines and vaccines), agriculture, the environment, and non-food industrial products. DNA can be modified spontaneously (by mutation) or artificially: humans can isolate, clone and transfer genes between different species. The main tools are bacterial enzymes, vectors (plasmids and viruses) and yeasts.

RESTRICTION ENZYMES AND STICKY ENDS

Restriction enzymes are enzymes that bind and cut DNA at specific base sequences. They were not invented: they are the natural defence system of bacteria, evolved to cut up and digest foreign DNA before it can integrate into the bacterial genome. The first restriction enzyme isolated from E. coli, EcoRI, cuts the sugar-phosphate backbone wherever it finds the sequence GAATTC, snipping between the G and the A on both strands. The staggered cut leaves single-stranded overhangs of exposed bases called sticky ends - ideal for binding to other DNA cut with the same enzyme.

EcoRI's staggered cut through GAATTC leaves matching single-stranded AATT overhangs on both fragments.

Not all restriction enzymes cut in a staggered way. Some cut straight across, leaving blunt ends: with no overhang there is no base pairing to satisfy, so any two blunt-ended molecules can be joined. In either case, the enzyme DNA ligase repairs the sugar-phosphate backbone and locks the joined fragments together.

GENE CLONING AND RECOMBINANT BACTERIA

Gene cloning produces large numbers of copies of a desirable gene. The steps are:

1. Locate the target gene with a DNA probe and cut it out with restriction enzymes.
2. Cut a bacterial plasmid (a small circle of bacterial DNA) with the same restriction enzyme, so the sticky ends match.
3. Mix gene and plasmid: the complementary sticky ends join, and DNA ligase seals the backbone. The result is a recombinant plasmid (recombinant DNA is DNA assembled from two different sources).
4. Insert the plasmid into bacteria by transformation.
5. As the bacteria multiply by binary fission, the gene is replicated with them - and expressed, so the protein can be harvested. One commercial use of gene cloning is the mass production of human insulin.

A gene cut with a restriction enzyme is pasted into a plasmid vector cut with the same enzyme.

How are the bacteria that actually took up the plasmid identified? An antibiotic-resistance gene is built into the plasmid, and the culture is grown on antibiotic-laced agar: every cell that failed to transform is killed, leaving only the recombinant cells.

Finding one gene among three billion bases requires a probe: a short (1-100 base), single-stranded fragment of DNA or RNA, labelled radioactively or fluorescently (read under X-ray or UV light), that base-pairs with its target sequence. Probes are used to screen libraries of recombinant bacteria for the clone carrying the gene. To design one, scientists work backwards from the protein's amino-acid sequence using the codon table - or borrow the gene's sequence from another species.

Different combinations of these tools achieve different goals:

• Selecting and removing a specific gene - a DNA probe, then restriction enzymes.
• Transgenesis (transferring a desirable gene from one organism to another) - select and remove the gene, then either insert it directly into the target cell (microinjection or a microparticle gun) or carry it in on a vector (a plasmid or virus cut with the same restriction enzyme).
• Gene cloning (producing many copies of a gene) - incorporate the gene into a bacterial plasmid and let the bacteria replicate.

GETTING DNA INTO CELLS

A viral vector's journey: the recombinant virus attaches to a receptor on the host cell, enters, travels to the nucleus, and its cargo gene integrates into the host genome - where, if the landing site is suitable, it is transcribed and translated like any native gene. Gene therapy uses exactly this route to correct a mutated or missing gene.

YEASTS

Bacteria cannot splice, so any gene given to them must first be trimmed to exons only. Yeast is a unicellular eukaryote: its plasmids can carry the entire gene, introns included, because yeast has the machinery to splice pre-mRNA itself. Yeast cultures already produce the Hepatitis B vaccine antigen, human growth hormone and insulin - and even manufactured opiates (codeine, morphine), reducing reliance on poppy crops.

CRISPR-CAS9

CRISPR stands for Clustered Regularly Interspaced Short Palindromic Repeats; Cas9 is the CRISPR-associated protein 9. In nature this is a bacterial immune system: the cell stores fragments of bacteriophage DNA as repeats, and if the virus invades again, a complementary RNA copy of the stored sequence combines with Cas9 to recognise and cut the viral DNA to pieces.

In the laboratory, the stored repeats are replaced with a synthesised guide RNA, designed to be complementary to any chosen gene. The guide RNA leads Cas9 to that exact sequence, where Cas9 can cut the DNA, delete or add genetic material - or, with a helper enzyme, change individual bases without cutting at all. The package is delivered into cells by microinjection, gene gun, liposomes or electroporation. The result is gene editing that is fast, cheap, highly accurate and able to target multiple genes at once - including the design of new proteins.

The synthetic guide RNA base-pairs with its matching DNA sequence, positioning Cas9 to cut precisely there.

DESIGNING NEW PROTEINS

Because a protein's shape follows its amino-acid sequence, and the sequence follows the gene, engineers can now design proteins that have never existed - candidate therapeutic drugs, new food sources, and smarter pest control. The steps are:

1. Select the features the protein must have.
2. Use computer modelling to suggest a shape that delivers them.
3. Determine an amino-acid sequence that folds into that shape.
4. Synthesise the 'gene' for that sequence by PCR.
5. Produce the protein in a culture system and test whether it works.

ETHICS OF GENETIC MANIPULATION

An organism carrying a gene from another species is transgenic. Uses include bacteria and yeast that make human insulin, and pest- or drought-resistant crops. DNA manipulation has, however, raised serious concerns in the community about its possible impact - genetically modified foods and transgenic crops may have unforeseen effects on the ecosystem. The main arguments on each side:

Each application deserves its own risk assessment: the benefits are real, and so are the stakes.