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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