Biotechnology: Principles and Processes

In this chapter: principles of biotechnology; restriction enzymes and naming; gel electrophoresis; cloning vectors (ori, selectable markers, cloning sites, insertional inactivation); vectors for plants and animals; competent hosts; the steps of recombinant DNA technology including PCR; bioreactors; downstream processing.

What biotechnology means

The European Federation of Biotechnology (EFB) defines it as "the integration of natural science and organisms, cells, parts thereof, and molecular analogues for products and services". Modern biotechnology rests on two core techniques:

1. Genetic engineering: altering the chemistry of the genetic material (DNA or RNA) and introducing it into a host organism to change its phenotype.
2. Bioprocess engineering: maintaining a sterile (microbial-contamination-free) environment so that only the desired microbe or eukaryotic cell grows in large numbers to make products such as antibiotics, vaccines and enzymes.

A piece of foreign DNA introduced into an organism will not multiply on its own. It must be linked to an origin of replication (ori). This is why a plasmid or other vector is needed.

The first recombinant DNA was made in 1972 by Stanley Cohen and Herbert Boyer, who cut an antibiotic-resistance gene from a plasmid of Salmonella typhimurium and joined it to a plasmid of E. coli.

Tools of recombinant DNA technology

Restriction enzymes (molecular scissors)

• In 1963, two enzymes that restrict the growth of bacteriophage in E. coli were isolated: one added methyl groups to DNA, the other cut DNA. The latter was called a restriction endonuclease.
• The first restriction endonuclease was Hind II. It always cuts at a particular point by recognising a specific sequence of six base pairs (the recognition sequence).
• More than 900 restriction enzymes have been isolated from over 230 strains of bacteria.
• They belong to the nucleases: exonucleases remove nucleotides from the ends of DNA; endonucleases cut at specific positions within the DNA.

Palindromic sequences: each restriction endonuclease recognises a palindrome, a base-pair sequence that reads the same on both strands when read in the same orientation (5'→3'). Example: 5'–GAATTC–3' / 3'–CTTAAG–5' is recognised by EcoRI.

Restriction enzymes cut a little away from the centre of the palindrome, between the same two bases on opposite strands. This leaves single-stranded overhangs called sticky ends, which form hydrogen bonds with complementary cut ends. DNA ligase then joins them. Cutting the vector and the source DNA with the same enzyme gives matching sticky ends.

Separating and isolating DNA fragments: gel electrophoresis

• DNA is negatively charged, so fragments move towards the anode in an electric field.
• The matrix most commonly used is agarose, a natural polymer extracted from sea weeds.
• Fragments separate by size through the sieving effect of the gel: smaller fragments move farther.
• Separated DNA is stained with ethidium bromide and seen as bright orange bands under UV light.
• Bands are cut out of the gel and extracted: this is elution. The purified fragments are used to build recombinant DNA.

Cloning vectors

Plasmids and bacteriophages replicate inside bacterial cells independently of the chromosomal DNA. Because bacteriophages are present in very high numbers per cell, their genome has a very high copy number inside the bacterial cell; some plasmids have only one or two copies per cell, others 15 to 100. Features needed in a cloning vector:

1. Origin of replication (ori): where replication starts. Any DNA linked to it can replicate in the host. It also controls the copy number.
2. Selectable marker: helps identify and eliminate non-transformants and selectively allows transformants to grow. These are normally genes giving resistance to antibiotics such as ampicillin, chloramphenicol, tetracycline or kanamycin. Normal E. coli has no resistance to any of these.
3. Cloning sites: the vector should have very few, preferably single, recognition sites for the commonly used restriction enzymes. More than one site produces several fragments and complicates cloning.
4. Vectors for plants and animals (see below).

pBR322 is the standard E. coli cloning vector. It carries ori, rop (codes for proteins involved in plasmid replication), and two resistance genes: amp^R (ampicillin) and tet^R (tetracycline). Restriction sites include Hind III, EcoR I, BamH I, Sal I, Pvu II, Pst I and Cla I.

Insertional inactivation

• If foreign DNA is ligated at the BamH I site of tet^R, the recombinant plasmid loses tetracycline resistance but keeps ampicillin resistance. Transformants are first grown on ampicillin, then transferred to tetracycline: recombinants grow on ampicillin but not on tetracycline.
• This two-plate selection is cumbersome, so a better method uses β-galactosidase. The recombinant DNA is inserted in the coding sequence of this enzyme, inactivating it (insertional inactivation).
• On a chromogenic substrate, colonies without an insert give blue colour; colonies with an insert (recombinants) stay colourless (white).

Vectors for cloning genes in plants and animals

• Agrobacterium tumefaciens, a pathogen of several dicots, delivers a piece of DNA called T-DNA into plant cells, transforming them into tumour cells. Its tumour-inducing (Ti) plasmid has been modified into a cloning vector that is no longer pathogenic but still delivers genes of interest.
• Retroviruses in animals transform normal cells into cancerous cells; disarmed retroviruses are now used to deliver desirable genes into animal cells.

Competent host

DNA is a hydrophilic molecule and cannot pass through cell membranes by itself. It can be introduced in these ways:

• Chemical: treat bacterial cells with a specific concentration of a divalent cation such as calcium, which increases the efficiency of DNA entry through pores in the cell wall. Cells with recombinant DNA are incubated on ice, given a brief heat shock at 42°C, then put back on ice.
• Micro-injection: recombinant DNA injected directly into the nucleus of an animal cell.
• Biolistics (gene gun): cells bombarded with high-velocity micro-particles of gold or tungsten coated with DNA; suitable for plants.
• Disarmed pathogen vectors: allowed to infect the cell and transfer the recombinant DNA into the host.

Processes of recombinant DNA technology

1. Isolation of DNA

Cells are broken open with enzymes: lysozyme (bacteria), cellulase (plant cells), chitinase (fungus). RNA is removed with ribonuclease; proteins with protease. Purified DNA precipitates as fine threads on adding chilled ethanol and can be removed by spooling.

2. Cutting DNA at specific locations

Purified DNA is incubated with the restriction enzyme under optimal conditions; digestion is checked by agarose gel electrophoresis. The vector is cut with the same enzyme.

3. Amplification of the gene of interest: PCR

• PCR (polymerase chain reaction) makes multiple copies of a gene in vitro.
• Needs two sets of primers (small, chemically synthesised oligonucleotides complementary to regions of DNA) and DNA polymerase.
• Three steps per cycle: denaturation (high temperature separates the strands), annealing (primers bind), extension (polymerase extends the primers).
• The enzyme is a thermostable DNA polymerase isolated from the bacterium Thermus aquaticus (Taq polymerase), which stays active at the high temperature of denaturation.
• Repeated cycles amplify a DNA segment about one billion times (roughly 10⁹ copies).

4. Insertion of recombinant DNA into the host

The ligated DNA is introduced into competent host cells. For example, if it carries an ampicillin-resistance gene and is transferred into E. coli, the cells become resistant to ampicillin; only transformants grow on ampicillin plates. The resistance gene acts as a selectable marker.

5. Obtaining the foreign gene product

• A protein coded by a recombinant gene expressed in a heterologous host is a recombinant protein.
• Small volumes of culture yield little product, so cells are grown in large volumes: continuous culture removes used medium from one side while adding fresh medium from the other, keeping cells in the logarithmic (exponential) phase for longer.

Bioreactors

• Vessels in which raw materials are converted into specific products by microbes, plant, animal or human cells. Volumes of 100 to 1000 litres are common.
• Provide optimal conditions: temperature, pH, substrate, salts, vitamins and oxygen.
• The most common is the stirring type: a simple stirred-tank bioreactor or a sparged stirred-tank bioreactor (air bubbled through). The curved base helps mixing; the stirrer mixes and makes oxygen available throughout; sparging increases the oxygen transfer area.
• Components: an agitator system, an oxygen delivery system, a foam control system, a temperature control system, a pH control system and sampling ports.

Downstream processing

After biosynthesis the product must be made ready for market: separation and purification, together called downstream processing. The product is then formulated with suitable preservatives. For drugs, it must undergo thorough clinical trials, and strict quality control testing is required for each product.

Common traps: (1) The first restriction endonuclease was Hind II, but EcoRI is the one whose naming is asked. (2) In EcoRI, "R" is the strain (RY 13), not "restriction". (3) Smaller fragments move farther in the gel; DNA moves to the anode. (4) Blue colonies = no insert; white colonies = recombinant. (5) Insertion at BamH I inactivates tet^R, so recombinants die on tetracycline, not ampicillin. (6) Biolistics is for plants; micro-injection for animal cells.

NEET focus

• Naming of restriction enzymes; palindromes; sticky ends; ligase.
• Gel electrophoresis details: agarose, ethidium bromide, UV, elution.
• Features of a vector; pBR322 map; insertional inactivation (tet^R and β-galactosidase).
• Ti plasmid, disarmed retrovirus, methods of making hosts competent.
• Enzymes used to break cells; PCR steps and Taq polymerase.
• Stirred-tank bioreactors and downstream processing.

Practice questions