Transcript GeneticEngineering4E.. - Bioinformatics Core Facility

Genetic Engineering for Engineers: A Brief
Introduction to Molecular Biology for Non-Biologist
Patricia Ayoubi, Ph.D.
OSU Microarray Core Facility
Department of Biochemistry and Molecular Biology
Oklahoma State University
Comparison between
Computer Systems and Living Organisms
Computer
System
vs.
Living
System
Information
Storage
Hard drive
(Disk - Applications)
Chromosomes
(DNA - Genes)
Stored
Information
Hard drive
(Applications)
Chromosomes
(Genes)
Launch
Application
(or open a file)
Transcription
of Genes
Information
Access
RAM
mRNA
An integrated circuit memory chip serving
as a temporary storage area in your PC
which stores information you are currently
working on.
A complex molecule serving as a
temporary storage area for the transfer of
genetic information from the cellular DNA
to other parts of the cell.
Accessed
Information
RAM
mRNA
Display
Translation
Information
Display
GUI
Proteins
Large numbers of
information
and functions
Complete
Systems
DNA (genes)
(hard drive - programs)
Chromosome
Transcription
mRNA
(RAM)
Translation
Protein
(GUI)
CENTRAL DOGMA OF INFORMATION FLOW
Essential components required for the CENTRAL DOGMA to function for
living systems include: polymerases, activators, transcription factors,
ribosomes, nucleotides, amino acids, tRNA, rRNA, enzymes, etc
Transcription
DNA (genes)
Translation
Protein
mRNA
(hard drive - applications)
(RAM)
Launch
(GUI)
Display
Essential components of computer systems to function include:
platters, spindle motor, heads, head actuator for the drive, CPU,
integrated circuits, transistors, power supply, etc.
The Underlying Code
Binary
Code
Genetic
Code
2 digits read eight at a
time = 28 or 256
combinations
4 nucleotides read three at
a time = 43 or 64*
combinations
Binary code = ASCII character
01000001 = A
01000010 = B
01000011 = C
01000100 = D
01000101 = E
Codon = amino acid
TTT = F
TCT = S
TAT = Y
TGT = C
CCT = L
*20 amino acids and 64 codons = redundancy
Genetic Engineering and
Recombinant DNA Technology
• DNA is structurally simple
enough to be manipulated
• Bio-molecules can act on and
react with other bio-molecules
• DNA can be very specifically
manipulated in test tubes
• DNA is a negatively charged
molecule (affected by electrical
current)
• Manipulated (engineered) DNA
can be put back into cells
• Engineered DNA can provide new
information for cells
• Engineered DNA can provide new
functions (new programs) to cells
How! is DNA manipulated…
Essential tools for genetic engineering
• Molecular scissors (restriction enzymes)
– cuts DNA at specific codes (sites)
•DNA Photocopiers (PCR)
– to replicate gene fragments on demand
•Molecular Glue (ligase)
– glues DNAs pieces cut with the same scissors
• Molecular “floppy disks” (plasmids)
– for temporarily storage of glued DNA
(glued DNA = recombinant DNA or rDNA)
• Cellular Factories (E. coli bacterium)
– to store and produce the rDNA on demand
Molecular Scissors cut DNA in specific places
Cut the DNA with
Molecular Scissors
“My Favorite Gene”
Note:
The specific DNA
piece carrying the
“My Favorite Gene”
program is typically
known (size) to
identify it in the mix.
Use agarose gel electrophoresis to isolate
the DNA fragment with “My Favorite Gene”
Extraction of “My Favorite Gene” from the gel
1.
Clean up sample and
remove gel matrix
2.
“My Favorite Gene”
is now ready for gluing
into “floppy disk”
(cloning)
Use DNA Photocopier to get “My Favorite Gene”
DNA Photocopier = PCR or
Polymerase Chain Reaction
http://www.dnalc.org/shockwave/pcranwhole.html
• Clean up sample
• “My Favorite
Gene” ready for
cloning
Molecular Glue to put DNA onto “floppy disk”
“My Favorite
Gene” frag
“floppy disk”
plasmid
Add molecular glue
(ligase)
rDNA
Work horse
Cells (E. coli)
Put into cells for safe
keeping and
propagation
Bacterial cells containing
Recombinant DNA = Clones
With Genetic Engineering and
Recombinant DNA Technology you can…..
• Clone genes into other simpler or model
organisms for study*
• Alter genes to disable protein activity
• Alter genes to enhance protein activity
• Completely remove a gene from an organism
• Add one or more novel genes to an organism
• Combine two or more genes to create a
multifunctional protein with unique activities
•Engineer novel metabolic pathways
* Most frequently used application
Engineering an existing pathway
Very Fast Process…
Very Slow Process…
Phenol (pollutant)
Phenol (pollutant)
X
…phenol accumulates and cell dies
Bacterium “a”
Phenol
Hydroxylase
Phenol
CO2 + water (harmless)
Phenol
Hydroxylase
CO2 + water
…cell goes hungry
Bacterium “A”
Cell now indulges on phenol!
Improved Bacterium “a”
Engineering a novel pathway
Sugar in…
Acid in…
…alcohol out
Bacterium B
Sugar in…
…acid out
+
…alcohol out
Bacterium A
$uperBug
Cut with a
restriction enzyme
Join to plasmid
with ligase
Produce a library
of clones