bacterial transformation

Bacterial transformation

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)

the genetic code is universal

In addition to the nucleoid DNA, E. coli bacteria contain small circles of DNA called plasmids;

plasmids

http://en.wikipedia.org/wiki/Plasmid

A plasmid is a small, circular piece of double-stranded DNA

plasmids

Often, the genes carried in plasmids provide bacteria with genetic advantages, such as antibiotic resistance.

Plasmid DNA contains coding sequences (genes) which are expressed by the bacterium (the bacterium produces the corresponding proteins ;

The cell that receives the piece of DNA (plasmid) is called transformed cell

Bacterial Transformation:

The introduction of a piece of DNA, like a plasmid, into a bacterial cell

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

Competent cells

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)

Bacterium without plasmid

Bacterial DNA

Bacterium with plasmid

(plasmids contain gene for antibiotic resistance)

In the presence of antibiotic ……..

In the presence of antibiotic ……..

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)

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.

Beta-lactam ring

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.

The ampicillin (penicillin)-resistance gene

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

The beta-galactosidase gene (sometimes called lacZ gene) encodes a protein, called beta-galactosidase;

This is an enzyme that normally cleaves the disaccharide sugar lactose into its two constituent sugars, galactose and glucose.

Color marker gene: beta-galactosidase

lactose galactose + glucose Beta-galactosidase

Plasmid containing beta-galactosidase gene

Color marker gene = beta-galactosidase

However, beta-galactosidase can also cleave a synthetic

analog of lactose called X-gal;

X-gal is colorless, but when it is cleaved by beta-

galactosidase, one of the products is dark blue;

Color marker gene: beta-galactosidase

X-gal identify cells with beta-galactosidase

When bacteria expressing beta-galactosidase are grown on a agar plate containing X-gal, the enzyme digests X-gal and produces a blue compound;

The colonies will be bright blue

If the bacteria do not produce beta-galactosidase, the colonies will be white

Origin of replication

a gene for resistance to an antibiotic (ampr)

Color marker gene (beta-galactosidase)

a sequence called polylinker

pBLU