Modeling Modeling TransformatTransformat

ionionWhat does each What does each

step do?step do?

Transformation Transformation ProcedureProcedure

Transformation Transformation ProcedureProcedure

Building Your ModelBuilding Your Model

Yarn Yarn = chromosomal DNA= chromosomal DNABeads Beads = ribosomes= ribosomesBlack Black velcro = plasmid DNAvelcro = plasmid DNAGreen Green velcro = GFP genevelcro = GFP genePink Pink velcro = Ampicillin resistance velcro = Ampicillin resistance

genegeneBottle = cell membraneBottle = cell membranePipe cleaner = flagellumPipe cleaner = flagellumCup = micro-tubeCup = micro-tube

pGLO PlasmidpGLO Plasmid

GFPGFP: : gene for gene for Green Fluorescent Green Fluorescent ProteinProtein

araCaraC: : gene for gene for Beta-lactamase Beta-lactamase (Ampicillin (Ampicillin resistance)resistance)

Real image of a bacterial plasmid (Scanning electron micrograph)

GFP

araC

E. coli E. coli CellCell

Bacterial chromosomal DNA

Cell membrane

-

-

---

-

-

- - - -

- - -Ribosomes

Add transformation Add transformation solutionsolution

Tube

CaCl2

Transformation Transformation solution: CaClsolution: CaCl22

Positive charge of CaPositive charge of Ca++++ ions shields negative ions shields negative charge of DNA charge of DNA

OCH2

O

P O

O

OBase

CH2

O

P

O

O

O

Base

OH

Sugar

Sugar

O

Ca++

Ca++

Ca++

Inoculate with bacteriaInoculate with bacteria

Tube

CaCl2

Add plasmidAdd plasmid

Tube

CaCl2

Incubate on iceIncubate on ice

Slows movement of cell Slows movement of cell membranemembrane

pGLO plasmids

Bacterial chromosomal DNA

Cell membrane

Heat-shock @ 42°CHeat-shock @ 42°C

Increases Increases permeabilitypermeability of of membranemembrane

pGLO plasmids

Bacterial chromosomal DNA

Cell membrane

Incubate on iceIncubate on ice

Returns bacteria to temperature Returns bacteria to temperature necessary for survivalnecessary for survival

pGLO plasmids

Bacterial chromosomal DNA

Cell membrane

Add nutrient broth Add nutrient broth (LB)(LB)

Provides nutrition for growth Provides nutrition for growth and gene expressionand gene expression

pGLO plasmids

Bacterial chromosomal DNA

GFP

Beta lactamase(ampicillin resistance)

What do you expect to What do you expect to happen?happen?

What do you expect to What do you expect to happen?happen?

What do you expect to What do you expect to happen?happen?

What do you expect to What do you expect to happen?happen?

What do you expect to What do you expect to happen?happen?

-pGLO

+pGLO

LB LB/amp

LB/amp

LB/amp/

arabinose