Genetic Engineering - E.coli

butterbeanscubaBiotechnology

Dec 12, 2012 (4 years and 8 months ago)

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Lecture 8


Genetic Engineering




Medically important substances
produced by genetic engineering


Human Insulin
-

used to treat diabetes


Past
: extracted insulin from pancreas of
cattle and pigs


Today
: Human insulin produced from
bacteria


Medically important substances
produced by genetic engineering


Vaccines:
genetically engineered
microorganisms used to make vaccines


Hepatitis B vaccine:
gene coding for viral
protein in Hepatitis B cloned into bacteria


Bacteria make viral protein


Protein used to make vaccine

Importance of Genetic Engineering


Genetic traits transferred to plants


Pest resistant plants:
engineered to resist
natural pests


Examples: corn, cotton, potatoes


Bt
-
toxin
: made in
B. thuringiensis


Toxic to insects
-

dissolves their gut


Insert gene that makes Bt
-
toxin into plants’


Plants now make Bt
-
toxin



Transgenic
: plants and animals into which new
DNA has been introduced


Figure 9.5

Table 9.2

Genetic Engineering


Transferring genes from one organism to
another organism


Can be prokaryote to prokaryote


Prokaryote to eukaryote


Or eukaryote to prokaryote


Requires several things:


DNA of interest (must be isolated)


Various enzymes (restriction enzymes)


Suitable vector


Method for introducing DNA into new host

Isolating DNA of interest

1.
Cells are lysed (burst open)

2.
As cells burst DNA is sheared into many
pieces of varying lengths

3.
Cut purified DNA into smaller fragments
using
restriction enzymes

Restriction Enzymes


When DNA is cut with restriction enzymes
the DNA pieces generated either have
sticky (cohesive ends) or blunt ends


The cohesive ends will adhere to any
piece of DNA with complementary
sequence

Table 9.3

Vectors


After isolating DNA, need to insert into
vector


Vector
-

modified plasmid


Contains:


Origin of replication


Selectable marker, example: antibiotic
resistance


At least one restriction enzyme recognition
site

Figure 9.15

Generation of Recombinant Vector


Recombinant vector
: vector containing the
piece of DNA you isolated


Put piece of DNA you isolated with sticky
ends together with vector that is cut with
same restriction enzyme


Sticky ends of isolated DNA will join to
sticky ends of vector


Add
ligase

to glue isolated DNA to vector




Same restriction enzyme used to isolate DNA of
interest




Introducing recombinant vector into
new organism


Three steps:

1.
Select a suitable host

2.
Insert DNA into cells

3.
Select for transformants

Select a suitable host


Usually use
E.coli


E.coli
is easy to grow and much is known
about it’s biochemistry and genetics


Also have known phenotypes such as
sensitivity to certain antibiotics

Inserting the recombinant vector
into bacterial cells


Two methods:

1.
DNA
-
mediated transformation


In this process competent cells take up DNA
from surrounding environment


2.
Electroporation

-

Process involves treating cells with electric
current that puts pores in the membrane so
that plasmid can enter

Selecting for bacteria that contain
vector


First, grow bacteria on
selective media
-

one that contains antibiotic


Remember, vectors contain genes for
certain antibiotic resistance


Introducing vectors into eukaryotic
cells


If introducing to plant cells, can use the
T
i

plasmid


Advantage of this is that T
-
DNA and any
genes you have inserted is naturally
transferred from Agrobacterium to host cell


Other methods:


Electroporation
: same as with bacterial cells


Gene gun
: device that uses a gas pulse or
other mechanism to propel DNA
-
coated gold
particles into cell