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 betweenComputer Systems and Living Organisms

Comparison betweenComputer Systems and Living Organisms

ComputerSystem

LivingSystem

vs.

InformationStorage

Hard drive (Disk - Applications)

Chromosomes(DNA - Genes)

StoredStoredInformationInformation

InformationInformationAccessAccess

Launch Application

(or open a file)

Transcription of Genes

RAMRAM mRNAmRNA

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.

AccessedInformation

RAM mRNA

InformationDisplay

Display Translation

GUI Proteins

Large numbers ofinformation

and functions

CompleteSystems

mRNA(RAM)

TranslationProtein

(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 FLOWCENTRAL DOGMA OF INFORMATION FLOW

The Underlying Code The Underlying Code

BinaryCode

2 digits read eight at a time = 28 or 256

combinations

Binary code = ASCII character01000001 = A01000010 = B01000011 = C01000100 = D01000101 = E

GeneticCode

4 nucleotides read three at a time = 43 or 64*

combinations

Codon = amino acidTTT = FTCT = STAT = YTGT = CCCT = 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…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 placesMolecular 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 gelExtraction of “My Favorite Gene” from the gel

http://www.dnalc.org/shockwave/pcranwhole.html

• Clean up sample• “My Favorite Gene” ready for cloning

Use DNA Photocopier to get “My Favorite Gene”Use DNA Photocopier to get “My Favorite Gene”

DNA Photocopier = PCR orPolymerase Chain Reaction

Digest(Molecular Scissors)

IsolateChromosomal DNA

Add to Vector(Molecular Glue)

Place into host cells

Work horseCells (E. coli)

rDNA

“My FavoriteGene” 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”Molecular Glue to put DNA onto “floppy disk”

Add molecular glue(ligase)

With Genetic Engineering andRecombinant DNA Technology you can…..

With Genetic Engineering andRecombinant 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)

PhenolHydroxylase

X

…cell goes hungry

Cell now indulges on phenol!

Improved Bacterium “a”

Phenol CO2 + waterPhenol

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 plasmidwith ligase

Produce a libraryof clones