Exploring the World of Microbiology & DNA Recombinant Technology





The microbiology department of HKU is solid and an informative branch. I was enlightened by the second lecturer of the Microbial Biotechnology course today, and wanted to share what microbiologists do in their lab on a daily basis.

The human ancestors dated back 2 million years ago. Microbes are found to have existed since 3.5 million years ago. Microorganisms constitutes as half of living organisms, which includes animals, plants, protozoa and fungi. Up to date, only 1% of bacteria species are identified and studied in the laboratory.

As more governmental funding are given to the field biotechnology, the development of many branches are as follows:

1. Medical diagnostic tests. Microbes are industrially applied in the recombinant DNA technology, which involves the transfer of target genes from one organism to another. This process can be used for the development of new vaccines and medicines. The protein products on the medicine market are produced from animal sources. The quintennsence example of microbial biotechnology is insulin. Industrial production of insulin comes from pigs and cows. In the past, natural souces insulin was collected in small amounts from these livestock and was sold very expensive. There is also the chance of microbial contamination from the livestock and must undergo purification to exclude other protein byproducts and surrounding microbial/viral contaminants. But now thanks to industrial technology, larger amounts of insulin are synthesized in the lab and made available for diabetes treatment.

Genentech is considered the founder company of the biotechnology industry.

Proprietary market of medicines are sold under brand names. Example: Aspirin is the brand name of acetaminophen.

2. Biotechnology food. Enzymes are used to process leather, wine, beer, yogurt. Feed enzymes are used for feeding livestocks. The Denmark company, Novozyme, is the largest company of industrial enzyme market.

3. Environmental biotechnology. The cleanup of natural disasters and hazardous waste spills. Organic fuel like gas and diesel can also be produced.

4. Biopesticides.

5. Industrial biotechnology.

6. DNA fingerprinting. The most elegant application of microbial biotechnology is the fingerprinting of DNA in forensic medicine. This branch of technology also allows for the parental testing where parents and childen relationship is identified by profiling their DNA.

In recombinant protein technology, the benefit of manufacturing synthetic penicillin is that they can be modified to confer resistance to the defense mechanisms of bacteria to the drug. Another instance, recombinant yeast far triumphs the natural baker’s yeast in all aspects of associated with its functions.

Where are biodegradable plastic found on campus? The biodegradable plastics are used for packaging take-out food.

The pharmaceutical industry is continually growing with the support of an increase growth of markets and development of R&D models. The drug market is undergoing an annual growth of 12% and spreads to more and more countries. As synthetic drugs are in higher demand, more and more projects arise in research and development.

Examples: Amgen, Genentech, Biogen Idec, Cephalon, Medimmune are American biotechnology companies that invest billions of dollars in the R&D of medicinal drugs. These companies hold large market shares and rack up large amounts of revenue. The two big players are Amgen and Genentech invests $3.4 billion and $3.0 billion, respectively. Hong Kong government funds for all Hong Kong universities with only $0.053 billion. Thus, America supplies more funding for pharmacetical companies than Hong Kong.

I now know the typical day is like the field of biotechnology, microbiology or pharmaceutical industry. They frequently apply DNA expression systems into their work.

What is empression system? Host, vector and DNA are the main players. Other contributors are plasmids, viruses (lambda bacteriophage), bacteria (E. coli), yeast (S. cerevisiae) and eukaryotic cell lines.

To a non-science major, the thought of DNA recombinant technology may be perplexing. However, it is quite simple. The foundation of recombinant technology involves 6 steps:

1. Isolation of target gene. Genes can be purchased.

2. Insertion of gene into a vector. Examples of vectors are bacterial plasmids, viruses, or shortgun directly into host cells.

3. Transform the vector into host cells. The plasmid can reside freely in the cytosol (episome) or integrated the host chromosome. Vectors are transformed into host cell by electroporation, chemical treatment or heat shock. Electroporation efficiency is only 20-30% successful. This procedure involves a brief spark of electrical shock that enlarges the pores of the plasma membrane. In this short time frame, the plasmid can enter the host cell and the pores close up. Chemical treatment kits such as calcium chloride are allegedly claimed to have 95% success rate. Heat shock is stimulated by subjecting the host cells to a sudden increase in heat, recover the cells and incubate them for further growth.

4. Growth of cells. The promoter of the gene is the induced to begin the cloning process by heat stimulation, ions or alcohol. As a selective tool for growing only viable cells conferring the gene of interest, the host cells are grown on selective media that can specifically select for antibiotic resistant cells. For instance, E. coli can confer resistant to kanamycin antibiotics.

5. Isolation and purification of gene protect. Genes can be amplified within the hour by polymerase chain reaction.

6. Formulation of the gene

Following the same concept of why cDNA derived from eukaryotes are used in prokaryotes, three points shouldd be considered:

— Eukaryotic proteins do not fold properly in prokaryotic host cells. These proteins also accumulate as insoluble aggregates.

— Proteins can undergo post-translation modifications such as N- and O-glycosylation, acetylation, methylation and phosphorylation. These can interfere with its function in the bacterial cell.

— Make sure that the nucleic acids DNA and RNA are complimentary. There are 64 codons encoding 20 amino acids, so some codons are redundant. The frequency of certain codons may far exceed others, leading to the lesser expressed codons to not be as efficient.