E. coli


14 déc. 2012 (il y a 9 années et 3 mois)

379 vue(s)

Biotechnology and
Recombinant DNA

What is Biotechnology


Use of microorganisms, cells or cell
components to make a product

Recombinant DNA technology (rDNA)

Genetic engineering

Insertion of genes into cells that makes the
cells into “factories” to make products

Recombinant DNA

Putting a gene from one organism into


Human insulin gene into a bacteria to make

Hepatitis B gene into a yeast to make the
hepatitis B vaccine

How to make rDNA

Gene of interest is inserted into a

Vector is usually a plasmid that must be

Cells containing the vector with the gene
of interest then divide to from a CLONE of
identical cells

These clones can then be used to harvest
the gene or produce a product

Restriction enzymes

DNA cutting enzymes that are a key to the
development of rDNA technology

Discovered in the early 1970’s

Nobel Prize in 1978 to Arber, Nathans and
Smith for the discovery of these enzymes

Restriction enzymes cut DNA at specific
sites and allow for DNA to be “inserted”
into a cloning vector

“Sticky ends” are generated?

Restriction Enzymes


DNA molecules that can be used as transfer
vehicles to insert DNA into cells

Must be self
replicating and small enough to
work with outside the cell

Plasmids are common vectors

Often contain antibiotic resistance gene

Viral DNA is also used as a vector

Retroviruses, Adenoviruses, Herpesviruses

Larger amounts of DNA can be inserted

Cloning plasmid

Kary Mullis

Invented the
technique of
polymerase chain
reaction in the early

Nobel Prize in 1993

Key technique used
to make large
quantities of DNA

Polymerase chain reaction

Inserting DNA into cells

1. Transformation

2. Electroporation

Electric current make pores in the cell so DNA
can enter

3. Protoplast fusion

Cells with no cell wall can be fused and
natural recombination may occur

4. Microinjection

Protoplast fusion

How are genes isolated?

1. Gene libraries

Digestion of entire genome with restriction

Insert fragments into vectors and put the
vectors into bacterial cells

2. Complementary DNA (cDNA)

Eukaryotic gene derived from mRNA made
with reverse transcriptase

Lacks introns only exons

Gene Library

Complementary DNA

Selecting the clone

Need to next be able to find the cell with the
gene of interest

Selecting the clone of interest is often done with
marker genes

Genes are spliced into plasmids carrying genes
for ampicillin resistance and

Colonies that grow with special characteristics
are selected as potential clones with the gene
you want!

Selecting recombinant bacteria

Selecting the clone

After candidate colonies are identified the
one with the gene of interest must be

DNA probes

Pieces of single stranded DNA
complementary to the desired gene are
made and labeled with a radioactive

Helps identify the target gene

DNA Probes

Making a gene product

Put the gene into a bacteria like
E. coli
and get
the product made

Toxic by
products from Gram

cell wall

Usually no secretion of product by Gram


Yeast cells

Better secretion of product

Mammalian cells

Good source for protein products

Little risk of toxins and allergy

Products of genetic engineering

Products of genetic engineering