STB1073 Biotechnology: Gene Cloning

Modern Biotechnology: Multidisciplinary Input

• Modern biotechnology involves the integration of biochemistry, genetics, microbiology, and cell biology.

General Strategy in Biotechnology

• The general strategy in biotechnology involves cloning a gene, inserting it into a suitable vector, transforming host cells, selecting transformed cells, culturing transformed clones, and extracting and purifying the recombinant product.

Cloning Vectors

• A cloning vector is a DNA molecule that carries a foreign DNA into a host cell (usually bacterial or yeast).
• Cloning vectors have the ability to replicate, producing many copies of themselves along with the foreign DNA.
• Types of cloning vectors include plasmids, phages, cosmids, bacterial artificial chromosomes (BAC), and yeast artificial chromosomes (YAC).

Plasmids

• Plasmids are small, circular, extrachromosomal DNA molecules found in bacteria that can replicate on their own outside of a host cell.
• Plasmids have a cloning limit of 100 to 10,000 base pairs or 0.1-10 kilobases (kb).
• Plasmids can confer advantages to host cells, such as antibiotic resistance, antibiotic production, sugar fermentation, degradation of aromatic compounds, heavy metal resistance, and toxin production.

Phages

• Phages are derivatives of bacteriophage lambda (λ phage), a virus that infects E. coli.
• Phages have a linear DNA molecule that can be replaced with foreign DNA without disrupting its life cycle.
• Phages have a high transformation efficiency, about 1000 times greater than that of plasmid vectors.
• Phages have a larger cloning limit than plasmids, consisting of 8-25 kb.

Cosmids

• Cosmids are extrachromosomal circular DNA molecules that combine features of plasmids and phages.
• Cosmids have a high transformation efficiency and a larger cloning limit than plasmids or phages, consisting of 35-50 kb.

Bacterial Artificial Chromosomes (BAC)

• BACs are based on bacterial plasmids and have a cloning limit of 75-300 kb.
• Examples of BACs include F-plasmid and P1-plasmid.

Yeast Artificial Chromosomes (YAC)

• YACs are artificial chromosomes that replicate in yeast cells.
• YACs are hybrids of bacterial plasmid DNA and yeast DNA.
• YACs have a cloning limit of 100-1000 kb, but have very low efficiency.

Microorganisms as Expression Hosts

• Microorganisms are used as expression hosts for the production of recombinant products, such as proteins, enzymes, and products of multi-enzyme pathways.
• Host requirements include suitable vectors, effective transformation, and ready fermentation and scale-up capacity.

Microbial Expression Hosts

• Examples of microbial expression hosts include Escherichia coli (E. coli), Saccharomyces cerevisiae, and Bacillus spp.
• E. coli has advantages such as numerous specific vectors, high transformation efficiencies, and well-understood fermentations, but has disadvantages such as low expression yields.
• Saccharomyces cerevisiae has advantages such as moderate expression yields and well-understood fermentations, but has disadvantages such as low-medium transformation efficiency and limited range of vector systems.
• Bacillus spp. has advantages such as high transformation efficiencies, high expression yields, and simple fermentations, but has disadvantages such as limited range of vector systems.

Expression Development

• Expression development involves the use of multicopy plasmids, multiple selective markers, different promoters, multiple promoters, multiple ori sequences, and N-terminal tags.

Viral Vectors

• Viral vectors are viruses that carry a modified or foreign gene and are commonly used in gene therapy.
• Examples of viruses used as vectors in gene therapy include retroviruses, adenoviruses, parvoviruses, herpesviruses, and poxviruses.

Contribution of Microbiology to Biotechnology

• Microbiology contributes to biotechnology by supplying antibiotics, cloning process, industrial enzymes, industrial products, food and beverages industries, research enzymes, medicine, and agriculture.

Agrobacterium in Biotechnology

• Agrobacterium is a bacteria that causes tumours in plants and has the ability to transfer genes to plants.
• The tumour-inducing (Ti) plasmid is responsible for the production of the tumour and the transforming abilities.
• The plasmid is 'disarmed' by deletion of the tumour-inducing genes, and the T-DNA is transferred and inserted into the host chromosome.

Golden Rice

• Golden rice is a genetically modified rice that produces beta-carotene, a precursor to vitamin A, to combat vitamin A deficiency.