Bacterial transformation
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Bacterial transformation. 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

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Bacterial transformation

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Bacterial transformation

Bacterial transformation


The genetic code is universal

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

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;


Plasmids1

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

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

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

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

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

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

Selection

Transformed cells contain the plasmid with ampicillin resistance gene

Non-transformed cells do not contain the plasmid

(agar plate)


How ampicillin works

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

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 (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

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

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

Plasmid containing beta-galactosidase gene

Color marker gene = beta-galactosidase


Color marker gene beta galactosidase1

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

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


Bacterial transformation

pBLU

  • Origin of replication

  • a gene for resistance to an antibiotic (ampr)

  • Color marker gene (beta-galactosidase)

  • a sequence called polylinker


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