Biomed Lab I FINAL

Prokaryotes

organisms made up of cells without a nucleus or any membrane-encased organelles

ex: bacteria

Plasmid

small, circular extrachromosomal DNA found in bacteria

Genome

the complete set of genetic information of an organism

Chromosomes

long strands of DNA molecules that store an organism’s genome

Genes

short segments of DNA containing protein-coding information

• located in chromosomal DNA

DNA

• aka deoxyribonucleic acid

• long chains composed of repeating subunits called nucleotides

  • Adenine, Tyrosine, Cytosine, Guanine

• antiparallel strands

  • DNA 5’ … 3’

  • RNA 3’ … 5’

pGLO Plasmid

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

GFP

a jellyfish gene that codes green fluorescent protein; responsible for green fluorescent phenotype

pBAD Promoter

a specific DNA sequence upstream from the GFP gene, which binds to araC-arabinose and promotes RNA polymerase binding and transcription of GFP

bla

a gene that encodes the enzyme beta-lactamase

• beta-lactamase breaks down the antibiotic ampicillin

responsible for antibiotic resistant phenotype

ori

the origin of pGLO plasmid DNA replication

araC gene

a gene that encodes the regulatory protein that binds to the pBAD promoter

only when arabinose binds to the araC protein is the production of GFP switched on

araC Protein

regulatory protein encoded by the araC gene

binds to the pBAD promoter

Arabinose

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

only when arabinose binds to araC protein is the production of GFP switched on

Ampicillin

an antibiotic that kills bacteria by destroying beta-lactamase

Recombinant DNA Technology

a method used to modify the gentic properties of an organism

useful in preparing proteins that are used in research and therapy

Steps of Recombinant DNA Technology

1) Synthesis of a recombinant plasmid

• genes are cut & recombined to create a plasmid

• plasmid must contain gene of interest, DNA for replication sequence, antibiotic resistance (may also have regulating & transcription gene

2) Transformation of the organism

• plasmid is inserted into bacteria cell

3) Selection of the transformed organism

• transformed cell is grown in antibiotic-containing media to allow plasmid to grow

4) Synthesizing the gene product (protein of interest)

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

Competent Cells

cells are capable of taking in foreign DNA

positive charge is used to treat cells to allow DNA to enter cell through cell membrane

Bacterial Transformation

method used to introduce plasmid DNA in bacteria

bacteria is made competent to take up foreign negatively charged DNA

QIAprep Spin Miniprep Kit

Buffer P1

Buffer P2

Buffer N3

Buffer PE

Distilled Water

Buffer P1

Resuspension Buffer

• removal of ions weakens cell membrane for lyse

• solution looks cloudy

Buffer P2

Alkaline Lysis Buffer

• ruptures cells and denatures DNA into single strands

  • solution looks clear to show lysis of cells & solubilation of cellular components

Buffer N3

Neutralization Buffer

• neutralization allows DNA to renature

• solution appears milky

Buffer PE

Wash Buffer

• ethanol helps wash chaotropic salts & other contaminants (buffer removed by centrifuge)

Distilled Water

water recovers “hydration shells” around plasmid DNA & dislodges plasmid DNA from silica gel membrane