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 Comparison between Computer Systems and Living Organisms

Computer System Living System vs. Information Storage Hard drive (Disk - Applications) Chromosomes (DNA - Genes)

StoredInformation InformationAccess Launch Application (or open a file) Transcription of Genes RAMmRNA Hard drive (Applications) Chromosomes (Genes) 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 Information Display Translation GUIProteins

Large numbers of information and functions Complete Systems

mRNA (RAM) Translation Protein (GUI) Transcription DNA (genes) (hard drive - programs) Chromosome

mRNA (RAM) Display Protein (GUI) Launch DNA (genes) (hard drive - applications) TranslationTranscription Essential components of computer systems to function include: platters, spindle motor, heads, head actuator for the drive, CPU, integrated circuits, transistors, power supply, etc. 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 CENTRAL DOGMA OF INFORMATION FLOW

The Underlying Code Binary Code 2 digits read eight at a time = 2 8 or 256 combinations Binary code = ASCII character = A = B = C = D = E Genetic Code 4 nucleotides read three at a time = 4 3 or 64* combinations 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 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

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” Molecular Scissors cut DNA in specific places

1.Clean up sample and remove gel matrix 2. “My Favorite Gene” is now ready for gluing into “floppy disk” (cloning) Extraction of “My Favorite Gene” from the gel

Clean up sample “My Favorite Gene” ready for cloning Use DNA Photocopier to get “My Favorite Gene” DNA Photocopier = PCR or Polymerase Chain Reaction

Work horse Cells (E. coli) rDNA “My Favorite Gene” frag “floppy disk” plasmid Bacterial cells containing Recombinant DNA = Clones Put into cells for safe keeping and propagation Molecular Glue to put DNA onto “floppy disk” Add molecular glue (ligase)

With Genetic Engineering and Recombinant DNA Technology you can….. 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 accumulates and cell dies Bacterium “A” Bacterium “a” Phenol (pollutant) CO2 + water (harmless) Phenol (pollutant) Phenol Hydroxylase X …cell goes hungry Cell now indulges on phenol! Improved Bacterium “a” PhenolCO2 + water Phenol Hydroxylase

Engineering a novel pathway Acid in… Sugar in… …acid out …alcohol out Bacterium A Bacterium B Sugar in… …alcohol out + $uperBug

Cut with a restriction enzyme Join to plasmid with ligase Produce a library of clones