Lecture (14) Biotechnology and bioremediation - Mt. San Antonio ...

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Feb 12, 2013 (4 years and 6 months ago)

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Biotechnology
By
Dr. Carmen Rexach
Microbiology
Mt 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 Technology
DNA & Protein biochips, biosensors, tissue &
organ cloning
Bioinformatics/Proteomics/Genomics
Pharmaceutical Biotechnology
vaccines, cancer treatments, gene therapy.
Agricultural Biotechnology
genetically enhanced crops & livestock
Environmental Sciences/Bioremediation
Neurobiology
Microbiology
Biomedical 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(-CH
3) of A and C
•palindromicsequences
•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
CTTAAG
DNA
Add restriction endonuclease
G
AATTC
G
CTTAA
Sticky ends
5’
3’
3’
5’
These sequences are
called 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
–CaCl
2
treatment
•Other methods
–gene guns/electroporation
–viral vectors
–mechanical insertion
Selection for transformed cells
Transformed
plasmid contains
antibiotic resistance
gene
Plate onto
agar containing antibiotic
Only those cells which have been transformed
will 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.
•Blumwaldand 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, Cais
using the tobacco plant as a production system for human
vaccines, cancer therapies (ex: lymphoma) and other
pharmaceuticals. “Pharmingin Plants”butno gene flow!
(Transient expression of mRNA of vaccine gene)
“Personalized medicine”
RNA
www.lsbc.com
Edible Vaccines
Introduce 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 Arntzenof 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
•Taqpolymerase
–DNA polymerase derived from Thermus
aquaticus
–works at very high temperatures
•dNTP’s
–deoxynucleotidetriphosphates
–provides nucleotides to build the new
strands
•Primers
–single-stranded
–delineate the DNA fragment to be amplified
•Thermal cycler
Thermal cycler
Amplification cycles
Denaturationof 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 agarosegel
+
-
Gel Electrophoresis: how it works
•Agarosegel 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 left
at 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
officials
detect 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
Dehalococcoidesand
dechlorinationof toxic compounds
•Dehalococcoidesethenogenescan
decontaminate 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 Dehalococcoidesspp.
•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 ½mile
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