the genetic code is universal All living things use the same genetic code Each codon corresponds to a specific amino acid, regardless of the species We can take a gene from one species and insert it into a different one and still get the same protein (same amino acid sequence)
plasmids A plasmid is a small, circular piece of double-stranded DNA http://en.wikipedia.org/wiki/Plasmid In addition to the nucleoid DNA, E. coli bacteria contain small circles of DNA called plasmids;
plasmids Plasmid DNA contains coding sequences (genes) which are expressed by the bacterium (the bacterium produces the corresponding proteins ; Often, the genes carried in plasmids provide bacteria with genetic advantages, such as antibiotic resistance.
Bacterial Transformation:The introduction of a piece of DNA, like a plasmid, into a bacterial cell The cell that receives the piece of DNA (plasmid) is called transformed cell
Competent cells Transformation rarely occurs naturally; By subjecting bacteria to certain artificial conditions, we can enable many of them to take up DNA; When bacterial cells are in a state in which they are able to take up DNA, they are referred to as competent
a plasmid contains: • an origin of replication • a gene for resistance to an antibiotic • Color marker gene • a sequence called polylinker(inside the coding sequence of the color marker gene) Color marker gene
Plasmid: origin of replication When a bacterium divides, all of the plasmids contained within the cell are copied; Each daughter cell receives a copy of each plasmid;
Plasmid: gene for antibiotic resistance This gene is useful to “select” the transformed cells (cells that contain the plasmid) Bacterial DNA In the presence of antibiotic …….. Bacterium without plasmid (plasmids contain gene for antibiotic resistance) In the presence of antibiotic …….. Bacterium with plasmid
Selection Transformed cells contain the plasmid with ampicillin resistance gene Non-transformed cells do not contain the plasmid (agar plate)
How ampicillin works? Ampicillin is a member of the penicillin family of antibiotics; Like other antibiotics, it works by keeping a bacterium from building a cell wall; Without the cell wall, the bacterium cannot live (the membrane bursts)
Beta-lactam ring Ampicillin (like other penicillin antibiotics) contains a chemical group called a beta-lactam ring; Bacteria build cell walls by linking molecules together: beta-lactams block this process.
The ampicillin (penicillin)-resistance gene The ampicillin-resistance gene encodes for a protein called beta-lactamase; This is an enzyme that destroys the activity of ampicillin by breaking down the beta-lactam ring.
Penicillin resistance Thus, bacteria expressing beta lactamase gene can resist the effects of ampicillin and other beta-lactam antibiotics (penicillin); These bacteria can grow in the presence of ampicillin
Color marker gene: beta-galactosidase Beta-galactosidase lactose galactose + glucose The beta-galactosidasegene (sometimes called lacZgene) encodes a protein, called beta-galactosidase; This is an enzyme that normally cleaves the disaccharide sugar lactose into its two constituent sugars, galactoseand glucose.
Plasmid containing beta-galactosidase gene Color marker gene = beta-galactosidase
Color marker gene: beta-galactosidase However, beta-galactosidasecan also cleave a synthetic analog of lactosecalled X-gal; X-gal is colorless, but when it is cleaved by beta-galactosidase, one of the products is dark blue;
X-gal identify cells with beta-galactosidase When bacteria expressing beta-galactosidaseare grown on a agar plate containing X-gal, the enzyme digests X-gal and produces a blue compound; The colonies will bebright blue If the bacteria do not produce beta-galactosidase, the colonies will be white
pBLU • Origin of replication • a gene for resistance to an antibiotic (ampr) • Color marker gene (beta-galactosidase) • a sequence called polylinker