Transcript Teaching Biotechnology, Brief History & Introduction to Recombinant

TEACHING BIOTECHNOLOGY
Philosophy:
In conveying the incredible power and
potential of biotechnology to students, it should
always be balanced with discussion of the Ethical,
Legal, Social/cultural Issues (E.L.S.I.) associated
with this technology.
Considering our past experience with new
innovative technologies, we must consider the long
term detrimental consequences as well as short
term solutions to problems.
LBNL ELSI Home Page
http://www.ornl.gov/sci/techresources/Human_Genome/elsi/elsi.shtml
As long as we keep this perspective,
biotechnology has the potential to address many
problems:
•Energy/Climate change
•Depletion of resources
•Pollution
•Disease/Health care
•Food production
How can biotechnology address these problems?
It requires a new paradigm in thinking about
technological solutions:
•Rather than learning about nature in order to
tame or overcome it, learn to mimic the way
nature does it!
BIOMIMICRY
•Energy production: mimic photosynthesis to create
voltage potentials, splitting H2O into H2 and O2
http://www.ls9.com/
•New materials that are biodegradable, no toxic wastes
•Air and water purification
•Food production??
A brief history of the scientific
discoveries leading to recombinant
DNA technology
1953 Watson & Crick discover the structure of DNA
How can scientists begin learning about the
structure and function of this HUGE molecule
(Genomic DNA)?
How can scientists begin learning about the
structure and function of this HUGE molecule
(Genomic DNA)?
•Need a way to break it down into bite-size pieces
•Need a way to amplify the bite-sized pieces so there is
enough to manipulate and study.
•In 1963 an enzyme was discovered in bacteria
that cuts double stranded DNA at a specific
nucleotide sequence
•Restriction Enzyme “molecular scissors”
Xba I
T▼CTAG A
A GATC▲T
HindIII
A▼AGCT T
T TCGA▲A
http://www.dnai.org/index.htm
•Shortly after restriction enzymes were discovered,
Kornberg discovered DNA Ligase during his investigation
of DNA replication.
•DNA Ligase forms phosphodiester bonds in the sugarphosphate backbone between DNA fragments
http://www.dnai.org/index.htm
Plasmids can be extracted out of bacteria,
manipulated, and introduced back into bacteria.
Replicate independently from genome to high copy
number.
Selection of recombinant plasmids by inactivation
of b-galactosidase gene expression
Jellyfish Aequorea victoria
Bacterial “Expression Vector” containing GFP gene
PLASMID MINI-PREPARATION
Procedure:
1. Concentrate cells: Spin down, resuspend in GTE.
2. Break cells open, denature DNA and proteins: Add SDS/NaOH
3. Neutralize NaOH, precipitate genomic DNA and protein: Add
Potassium Acetate/Acetic acid
4. Remove protein/DNA “snot”: Centrifuge, keep supernatent (liquid
portion) containing plasmid and RNA.
5. Concentrate nucleic acid: Isopropanol precipitation, ethanol wash.
6. Dry nucleic acid pellet