Biotechnology

ByDr. Carmen Rexach

MicrobiologyMt San Antonio College

What is biotechnology?

The use of living organisms, or parts thereof, to provide useful products, processes and services.

A Collection of Technologies that uses cells and biological molecules (DNA, RNA, proteins, etc.) to create new products, processes or services.Involved in: medicine, agriculture, engineering, law enforcement, environmental clean-up, energy production, basic life science research, etc.

Also includes….

Health Sciences & Medical TechnologyDNA & Protein biochips, biosensors, tissue & organ cloning

Bioinformatics/Proteomics/GenomicsPharmaceutical Biotechnology

vaccines, cancer treatments, gene therapy.Agricultural Biotechnology

genetically enhanced crops & livestockEnvironmental Sciences/Bioremediation Neurobiology MicrobiologyBiomedical Engineering, Nanotechnology, … & More!

Techniques for manipulating DNA

• Recombinant DNA• Polymerase chain reactions (PCR)• RFLP analysis• Gel electrophoresis• Southern blot

Recombinant DNA

• Occurs naturally– transformation– meiosis

• Technique in which a gene of interest is transferred into another genome

• Requires special enzymes– restriction endonucleases

• Requires competent cells

Restriction endonucleases

• Recognize specific sequences in DNA and cut it at or near a recognition sequence in a consistent way

• Naturally occurring bacterial defense mechanism against viral invasion– How is the bacterial DNA protected?

• by methylation (-CH3) of A and C

• palindromic sequences• sticky ends

Examples of restriction sites• G ^ AATTC• CTTAA ^ G

• A ^ AGCTT• TTCGA ^ A

• G ^ GATCC• CCTAG ^ G

Eco R1

Hind III

Bam H1

Recognition sequence

GAATTC

CTTAAGDNA

Add restriction endonuclease

G

AATTC G

CTTAA

Sticky ends

5’ 3’

3’ 5’

These sequences arecalled palindromes

Add new DNA fragment

CTTAA

G

G

AATTC AATTAGCAGC

TCGTCGTTAA

AATAAGCAGC

TTCGTCGTTAA

DNA fragment

Join with DNA ligase

Competent cells• Definition: cells that can take up DNA in solution• Recombinant DNA can be inserted into bacterial cells• Cells may be naturally competent or can be made

competent– CaCl2 treatment

• Other methods– gene guns/electroporation– viral vectors– mechanical insertion

Selection for transformed cells

Transformedplasmid containsantibiotic resistancegene

Plate ontoagar containing antibioticOnly those cells which have been transformedwill grow

Example: Insulin gene

Salt Tolerant Tomatoes May Hold the Key to Growing Crops in Marginal Soils (and may even reclaim salt from the topsoil)

• 24.7 million acres of once agriculturally productive land are being lost annually because of irrigation-induced salinity (USDA). • Crop production is limited by salinity on 40 % of the world's irrigated land and on 25 % of the land in the USA. • Blumwald and Zhang genetically engineered tomato plants that produce higher levels of a “sodium transport protein." Store salt in vacuole of leaves/roots• Plants grow and produce fruit even in irrigation water that is > 50X saltier than normal. Nature Biotechnology, Aug 2001

Blumwald & Zhang’s Work

Transgenic tomatoes in 5mM (A) and 200mM (B) of NaCl

2 & 3 Have gene for transporter

UC Davis Research:

Use Tobacco to make medicines

Large Scale Biology (Aka Biosource) in Vacaville, Ca is using the tobacco plant as a production system for human vaccines, cancer therapies (ex: lymphoma) and other pharmaceuticals. “Pharming in Plants” but no gene flow!

(Transient expression of mRNA of vaccine gene)

“Personalized medicine”

RNA

www.lsbc.com

Edible VaccinesIntroduce antigenic proteins from disease-causing organisms into plants. Eating the fruit or vegetable can then induce antibodies just like a vaccination, rendering the person immune to the disease.

The feasibility of this approach has already been demonstrated. Dr. Charles Arntzen of Arizona State University. He is actively pursuing research to allow children to be immunized against debilitating diseases such as hepatitis B, for example, by simply eating a modified banana, potato or tomato. Also Bill Langridge, Loma Linda Univ. in California. ( Sci. Am., Sept. 2000)

Polymerase chain reaction

• In vitro method of generating many copies of a specific DNA fragment

• Special requirements– Equipment

• Thermal cycler– Substrates and primers

• heat resistant DNA polymerase• source of nucleotides• DNA primers

Requirements

• Taq polymerase– DNA polymerase derived from Thermus

aquaticus– works at very high temperatures

• dNTP’s– deoxynucleotide triphosphates– provides nucleotides to build the new

strands

• Primers– single-stranded– delineate the DNA fragment to be amplified

• Thermal cycler

Thermal cycler

Amplification cycles

Denaturation of DNA

Hybridization of primers

Extension with dNTP’s

Double stranded DNA

(annealing)

RFLP: restriction fragment length polymorphisms

• Method of DNA fingerprinting• Premise: Many regions of DNA exist in the genome that

vary greatly between individuals = polymorphisms• How it works:

– digest homologous chromosomes with the same restriction enzymes

– or , amplify particular regions with PCR– Run the resulting fragments out on a gel (gel electrophoresis)

and compare the restriction bands • abundant in the genome• Great tool for forensics cases!• Analyze by Southern Blot

RFLP

Analysis

• Gel electrophoresis• Southern blot

Gel electrophoresis

• Separate DNA fragments on agarose gel

+-

Gel Electrophoresis: how it works

• Agarose gel prepared and placed in a gel box filled with buffer

• DNA is loaded into small pits at one end of gel

• Electrodes are placed at each end of gel box

• What is the charge of DNA?• Which electrode will it migrate

towards?• Movement through the gel

matrix separates out different sized fragments

Result: DNA fingerprint

Southern blot

• Purpose: To find a particular DNA sequence

1. Run a gel with DNA 2. Transfer to a membrane

3. Incubate with probe of interest

Uses of information from DNA

identify potential suspects whose DNA may match evidence leftat crime scenes

exonerate persons wrongly accused of crimes identify crime and catastrophe victims establish paternity and other family relationships identify endangered and protected species as an aid to wildlife

officialsdetect bacteria and other organisms that may pollute air, water,

soil, and food match organ donors with recipients in transplant programs determine pedigree for seed or livestock breeds

Bioremediation• Using bacteria to detoxify

or degrade pollutants• Use of bacterial enzymes

to remove clogs in drains• Most common bacteria

used:– Pseudomonas– Bacillus

Bioremediation

• May take advantage of natural processes– Ability of some microbes to metabolize

petroleum under aerobic conditions• May involve bioaugmentation

– Organisms selected for growth on specific nutrients

– Genetically engineered organisms

Removal of selenium

• Essential nutrients required in small amounts

• Present in high concentrations due to irrigation in California

• Certain bacteria can neutralize this substance

Dehalococcoides and dechlorination of toxic compounds

• Dehalococcoides ethenogenes candecontaminate tetrachloroethene (PCE), trichloroethene (TCE), and polychlorinated biphenyls (PCB)

• Utilize these substances as electron acceptors in anaerobic respiration

• Pathway• PCE TCE 1,2 Dichloroethene Vinyl chloride ethene

(environmentally benign)

•KB-1 is a commercial microbial culture that includes high numbers of Dehalococcoides spp.

•Bacteria will be introduced into 200 wells, 180 ft deep, in Seal Beach

•Purpose: to remediate a toxic plume 2/3 of a mile long and ½ mile wide threatening groundwater in Seal Beach.

•Expected outcome: Bacteria will create a biological barrier to prevent TCE contamination of groundwater

•Bioremediation should be complete in 14 years