Biotechnology Applications

• Biopharmaceuticals
  • Insulin
• Agriculture: Modifying Crops
  • Glyphosate Resistance
  • Nutritional Enhancement
• Forensics
  • STR Profiles
  • Cases
• Gene Editing
  • CRISPR

What is Biotechnology?

Biotechnology is “any technological application that uses biological systems, living organisms, or derivatives thereof, to make or modiy products or processes for specific use”.

Insulin

• First used in 1922 to treat Type I diabetes.
• Extracted and purified from pancrease glands of animals (cows, pigs, horses).
• Eli Lilly started large scale production of highly purified insulin in 1923
• By 1970, insulin production was large scale.
• By 1977, Lilly contracted competing institutions to use recombinant DNA technology to produce insulin.

Making Insulin: 1970s

• 8000 pounds of pancreas glands needed to make 1 pound of insulin.
• Required 56 million animals per year.

Cloning the Insulin Gene

• Insulin is a portein hormone with 51 amino acids on two different polypeptide chains.
• The chains are held together by disulfide bonds.
• Making eukaryotic proteins in bacterial hosts is difficult since bacteria doesn’t work well with eukaryotic proteins.

Biotechnology and Crop Plants

Usages:

• Herbicide Resistance
• Nutritional Enhancement
• Disease Resistance
• Cold Resistance

Gene for Glyphosate Resistance

• EPSP synthase, an enzyme important in amino acid biosynthesis, is cloned from resistance strain of E. coli. and the gene is transferred to corn and soybeans.
• High levels of EPSP synthase make cell resistant to glyphosate, a herbicide that inhibits EPSP synthase.

Nutritional Enhancement

• Vitamin A deficiency is the leading cause of childhold blindness in 118 countries.
• 140-180 million children are affected
• 500,000 go blind every year, die in 1 year.
• 600,000 women die from childborth complications, most of which are curable with Vitamin A.
• Golden Rice Project offers a partial solution to this problem.
  • Approach consists of introducing necessary enzymes to enable rice grains to process precursor molecules present in the grain.
  • This serves as a \(\beta\)-Carotene pathway, which is the vitamin A precursor.

Biotechnology in Forensics

• Forensics is the use of science and technology to investigate and establish facts in criminal or civil courts of law.
• Typically involves recovery and analysis of biological samples.
• Palo Verde tree case
  • Two seed pods found in bed of Mark Allen Bogan’s truck
  • Geneticist matched seeds to a palo verde tree, which was at the site where Denise Johnson was strangled.

STR DNA Profiling

STRs (short trandem repeats) are repeats of base pairs (2-9 in length) repeated 7-40 times. Each cluster is a heritable allele. Hundreds of STR loci are present in the human genome. FBI and other law enforcement selected 13 of the STR loci to be used as a set for forensic analysis. The markers are known as the CODIS panel (COmbined DNA Index System)

DNA Profiles: Methodology

• Each primer set is tagged with fluorescent dyes.
• After the DNA is amplified, there are large amounts of the fluorescently labeled primer sets.

Phylogenetic Analysis and HIV

• An AIDS patient with no known risk factors had an invasive procedure done by an HIV positive dentist.
• There were 6 others who were also infected.
• Envolopne genes from each patient and HIV positive controls were analyzed by:
  • amino acid signature
  • phylogenetic analysis
  • genetic distance measurements
• 5/6 patients were infected by the dentist
• There are shared evolutionary relationships in the DNA sequence. These are used in Phylogenetic Tree Analysis.

Gene Editing Systems

• Zinc finger nucleases (ZFNs)
  • Combines a zinc-finger DNA binding protein with a DNA-cutting enzyme. Each protein can then be synthesized to recognize the zinc-finger and cut the nuclease at specific sites
• TALENs
  • TAL subunits can be assembled to recognize any DNA sequence of choice. Coupled with a DNA-cutting enzyme to form TALEN. TALENs can then recognize and cut at specific sequences.
• CRISPR/Cas
  • CRISPR is an RNA with one end that binds to any DNA sequence of choice, the other binds to a DNA-cutting enzyme.
• In each system, DNA at cut sides can be modified, deleted, or replaced in specific ways.

CRISPR/Cas systems

• Help bacteria resist viral infection.
• Clusted Regular Interspaced Palindromic Repeat.
• Simple two component system
  • Guide RNA (sgRNA) with a target finding sequence and a duplex structure that binds to Cas.
  • Cas9 an endonuclease that makes double stranded cuts in DNA at target site.