Biotechnology: Principles and Processes

Two core techniques

• EFB (European Federation of Biotechnology) definition: "the integration of natural science and organisms, cells, parts thereof, and molecular analogues for products and services."

The 3 basic steps of genetic modification

1. Identify the DNA with the useful gene.
2. Introduce it into a host organism.
3. Keep the new DNA in the host and pass it on to the host's offspring.

The toolkit

• Restriction enzymes: "molecular scissors"
• DNA ligase: "molecular glue"
• Vectors: carriers such as plasmids and bacteriophages
• Competent host: e.g. E. coli

1. Restriction enzymes: molecular scissors

• They cut DNA at specific sequences.
• The first one discovered was Hind II. It always cuts at a particular 6 base-pair sequence.
• Exonucleases cut nucleotides from the ends of DNA. Endonucleases cut inside the DNA at specific points.

How they are named: EcoRI

Palindromes: restriction enzymes recognise palindromic sequences. These read the same on both strands in the 5'→3' direction (like the words "MADAM" or "RACECAR").

EcoRI site:   5' — G ↓A A T T C — 3'
              3' — C T T A A↑ G — 5'

After cutting:
              5' — G           A A T T C — 3'
              3' — C T T A A           G — 5'
                   └── sticky ends ──┘

• EcoRI cuts between G and A on each strand, leaving short single-stranded overhangs called sticky ends.
• Sticky ends easily stick to matching ends from the same enzyme. DNA ligase then seals them.

2. Gel electrophoresis: sorting DNA pieces by size

1. DNA pieces are placed in wells of an agarose gel.
2. An electric current is switched on. DNA (negative) moves towards the anode (+).
3. Small pieces move fast and far; big pieces move slowly.
4. The gel is stained with ethidium bromide and viewed under UV light → orange bands.
5. The band is cut out (elution) to get the pure DNA piece.

• Agarose comes from seaweed.
• DNA is negatively charged (because of the phosphate groups), so it moves towards the positive electrode (anode).

3. Cloning vectors: the delivery trucks

A vector carries the foreign gene into the host. Usually a plasmid (small circular DNA in bacteria) or a bacteriophage (a virus that infects bacteria).

What a good vector needs:

Finding the right cells: insertional inactivation

1. The foreign gene is inserted into the vector's lacZ gene, which breaks it (insertional inactivation).
2. Bacteria are grown on a plate with a chromogenic substrate.
3. White (colourless) colonies = recombinants (lacZ broken, no colour).
4. Blue colonies = non-recombinants (lacZ works and makes the blue colour).

Vectors for plants and animals

• Agrobacterium tumefaciens causes crown gall disease in plants. Its Ti plasmid is disarmed (made harmless) and used to carry useful genes into plants.
• Retroviruses can be disarmed and used to carry genes into animal cells.

4. Competent host: getting DNA into cells

DNA can't cross cell membranes on its own (it is hydrophilic), so we make cells "competent":

The complete process of recombinant DNA technology

1. Isolate DNA: break the cells with enzymes, remove RNA and protein, add chilled ethanol → DNA threads appear.
2. Cut DNA with restriction enzymes; check by gel electrophoresis.
3. Amplify the gene with PCR (make millions of copies).
4. Ligate: join the gene into a vector with DNA ligase.
5. Insert the recombinant DNA into a host; select the transformants.
6. Grow the host on a large scale in bioreactors → product.
7. Downstream processing: purify, add preservatives, test.

Step 1 detail: breaking open cells

• Then RNA is removed with ribonuclease and proteins with protease.
• Chilled ethanol makes the pure DNA come out as fine threads, which can be lifted out (spooling).

Step 3 detail: PCR, the DNA photocopier

PCR = Polymerase Chain Reaction. It makes millions of copies of a gene in a few hours. Each cycle has 3 steps:

1. Denaturation: heating (~94 °C) separates the DNA strands.
2. Annealing: on cooling, the primers bind.
3. Extension: Taq polymerase builds the new strands.
4. The cycle is repeated about 30 times.

• Taq polymerase comes from the bacterium Thermus aquaticus, which lives in hot springs. The enzyme doesn't get destroyed by heat.
• Primers are short DNA pieces that mark where to start copying.
• Copies double every cycle: after n cycles there are about 2ⁿ copies. About 30 cycles give about 1 billion copies!

Step 6 detail: bioreactors

• Bioreactors are large tanks (100–1000 litres) where the host cells grow and make the product.
• The most common type is the stirred-tank bioreactor. It has:
  • an agitator (stirrer) to mix,
  • an oxygen delivery system (sparger, which bubbles air),
  • a foam control system,
  • temperature and pH control,
  • a sampling port.
• Continuous culture: used medium is drained out and fresh medium added, which keeps the cells in their most active log (exponential) phase.

Step 7: downstream processing

• Separation and purification of the product.
• Formulation with preservatives.
• Clinical trials (for medicines) and quality control testing.

30-second revision

• First rDNA: Cohen and Boyer (1972), Salmonella typhimurium plasmid.
• EcoRI = E. coli RY13, first enzyme. Palindrome GAATTC, sticky ends.
• DNA runs to the anode; small pieces go farther; ethidium bromide + UV; elution.
• Vector needs ori, selectable marker, cloning sites. pBR322: ampR, tetR.
• Blue–white selection: white = recombinant (lacZ broken).
• Ti plasmid of Agrobacterium for plants; Ca²⁺ + heat shock for bacteria.
• PCR: denature → anneal → extend (Taq polymerase); 2ⁿ copies.
• Stirred-tank bioreactor; downstream processing.