Lab 6: Bacterial Transformation, Plasmid DNA Isolation

Prokaryotes

organisms made up of cells that lack a cell nucleus or any membrane-encased organelles, ex. bacteria

Plasmids

small, circular extrachromosomal DNA found in bacteria

Plasmid Replication

can replicate independently of chromosomes and generally result in phenotypic changes required for surviving in a hostile environment, ex. antibiotic resistance

Plasmid DNA Transfer

occurs naturally in bacteria through transformation

Genome

the complete set of genetic information, stored in chromosomes

Chromosomes

long DNA molecules that store the genome

Genes

short DNA segments containing protein coding information and are located in chromosomal DNA, also present on mitochondrial DNA in higher organisms

DNA

deoxyribonucleic acid, very long chains composed of nucleotides A, G, C, T, double stranded, present in eukaryotic mitochondria and bacterial plasmids

A

Adenine

G

Guanine

C

Cytosine

T

Thymine

Nucleotides

repeating subunits

DNA: Strands

complementary, A pairs with T, C pairs with G, antiparallel, twist to form a helix

DNA Helix

2 DNA strands, antiparallel strands, supercoiled

Antiparallel

opposite direction to each other

Extra-Chromosomal DNA

mitochondrial and plasmid DNA

pGLO Plasmid

recombinant, contains several genes and DNA sequences that enable replication of the plasmid DNA and differentiate and retransform bacteria

GFP

the jellyfish gene that codes green fluorescent protein and responsible for the green fluorescent phenotype

pBAD Promoter

specific DNA sequence from the GFP gene that binds araC-arabinose and promotes RNA polymerase binding and GFP transcription

bla

gene that encodes the enzyme beta-lactamase and responsible for the antibiotic resistant phenotype

Beta-Lactamase

breaks down the antibiotic ampicillin and allows bacteria to grow in presence of ampicillin

ori

the origin of pGLO plasmid DNA replication

araC Gene

gene that encodes the regulatory protein that binds to pBAD promoter

GFP Production

can only occur when arabinose binds to araC protein

araC Protein

the regulatory protein encoded by araC gene, binds to pBAD promoter

Arabinose

a sugar that binds to pBAD promoter and displaces araC protein from the promoter so RNA polymerase can bind and initiate transcription

Ampicillin

an antibiotic that kills bacteria, destroyed by beta-lactamase enzyme encoded by bla gene

Transformed Bacteria

survive/grow in the presence of ampicillin, can be selected from the non-transformed bacteria by growing in an environment containing ampicillin

Example of Transformed Bacteria: Neomycin Phosphotransferase II Gene Product

neomycin phosphotransferase II degrades neomycin, neomycin is used for selecting the bacteria transformed with the respective plasmid

Recombinant

DNA from one organism foreign to the bacteria or yeast is inserted into the bacteria’s plasmid

Recombinant Plasmid

DNA vector, transformed in cells that are grown to produce more plasmids or proteins

Recombinant DNA Technology Steps

synthesis of a recombinant plasmid, transformation of the organism, selection of the transformed bacteria, synthesizing the gene product/protein

Recombinant DNA Technology: Synthesis of a Recombinant Plasmid

• DNA fragments (genes) are cut and recombined to create a plasmid

• plasmid must have origin of replication sequence of DNA and an anti-biotic resistant gene for survival in antibiotic-containing media

• other genes for controlling transcription of gene products can be included

Recombinant DNA Technology: Transformation of the Organism

the plasmid is inserted in bacteria cell

Recombinant DNA Technology

the transformed cell is grown in an antibiotic containing media so that only bacteria cells containing the plasmid will grow and non-transformed cells will die

Recombinant DNA Technology: Synthesizing Gene Product/Protein of Interest

the cells are grown in media that contain molecules required for producing the protein

Competent Cells

cells treated with calcium chloride (CaCl₂) that are capable of taking in foreign DNA, cells kept frozen until use

Competent Cells: Calcium Effect

positive charge of calcium ions shields the negative charge of the DNA phosphates, makes it easier for DNA to enter cell through the cell membrane

Competent Cells: Glycerol

prevents formation of ice crystals inside the cells and increases their survival

E. coli Strain

derived from pathogenic E. coli, generally non-pathogenic

Competent Cells: Enzymes

unable to produce enzymes that metabolize arabinose, not ampicillin resistant, and do not have GFP because they do not contain pGLO plasmid

Competent Cells: no pGLO Plasmid

cannot grow on media containing ampicillin and exhibit green fluorescence

Competent Cells: L (+) arabinose

when L (+) arabinose is added, the transformed cells produce GFP and the transformed colonies appear green

Transformation

a method to introduce plasmid DNA in bacteria

Bacterial Transformation Process: Foreign DNA Uptake

to uptake the foreign DNA, the bacterial cells are made competent to receive the highly negatively charged DNA

Bacterial Transformation Process: Heat/Cold Shock

for heat shock, the transformation mixture is incubated on ice to make them cold, slowing the fluid cell membrane, then the mixture is quickly warmed up

Bacterial Transformation Process: Rapid Temperature Change Due to Heat/Cold Shock

shocks the cells and increases permeability of cell membrane

Bacterial Transformation Process: Nutrient Broth

helps recover shocked cells in cell membrane, often grown overnight on agar plates

Agar Plates

plates containing antibiotic, bacteria appear as translucent colonies on some

Bacteria Colony

represents a single transformed cell (clone)

LB Agar Plates Without Antibiotic

used as controls to demonstrate that the cells were alive and survived the transformation

LB Agar Plates with Antibiotic

used to select the transformed cells that have an antibiotic resistant phenotype and grow in presence of the antibiotic

RNase

an enzyme that rapidly degrades RNA, added to P1 buffer to exclude RNA in the DNA extract

P1 Buffer

resuspension buffer,

P2 Buffer

N3 Buffer

PE Buffer

Distilled Water