E. coli

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14 Δεκ 2012 (πριν από 4 χρόνια και 6 μήνες)

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Biotechnology and
Recombinant DNA

What is Biotechnology


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
another


Examples:



Human insulin gene into a bacteria to make
insulin


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

How to make rDNA


Gene of interest is inserted into a
VECTOR


Vector is usually a plasmid that must be
self
-
replicating


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

Vectors


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
1980


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
enzymes


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
β
-
galactosidase


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
selected


DNA probes


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


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
-

cells


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