Recombinant Technology

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14 Δεκ 2012 (πριν από 4 χρόνια και 9 μέρες)

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Recombinant DNA,
Biotechnology, and Microbes

Microbiology 221

Overview


Putting microbes to
Work


Molecular Cloning


Recombinant DNA technology

utilizes the power of
microbiological selection and screening procedures
to allow investigators to isolate a gene that
represents as little as 1 part in a million of the
genetic material in an organism.


The DNA from the organism of interest is divided
into small pieces that are then placed into individual
cells (usually bacterial).



These can then be separated as individual colonies
on plates, and they can be screened through rapidly
to find the gene of interest.

Recombinant DNA( natural and
manipulative)


Combination of DNA from organisms
from two different sources


Bacterial and human


Bacterial and plant


Viral and human

Basics of Restriction enzymes


Isolated from various bacteria,
restriction enzymes recognize short
DNA sequences and cut the DNA
molecules at those specific sites.


(A natural biological function of these
enzymes is to protect bacteria by
attacking viral and other foreign
DNA.)

Process



Restriction endonucleases

cut at defined sequences of
(usually) 4 or 6 bp. They cut on both strands of DNA



This allows the DNA of interest to be cut at specific
locations. The physiological function of restriction
endonucleases is to serve as part of system to protect
bacteria from invasion by viruses or other organisms


Cuts yield either "staggered" or "sticky" ends (see figure) or
"blunt" ends.


Two pieces of DNA cut with the same enzyme, can be pasted
together using another enzyme called "DNA ligase".

Sticky ends

Sticky ends


When the ends of the restriction
fragments are complementary,


EcoRI


recognition sequence


5'
---
G ‘AATTC
---
3'








3'
---
CTTAA ‘G
---
5'



Blunt ends



(1)

The restriction endonuclease
cleaves in the center of the
pseudopalindromic recognition site to
generate blunt (or flush) ends.




HaeIII


GG'CC



HincII


GTY'RAC

Restriction enzymes generate fragments that
facilitate recombination

Process


Cut ends in recognition sequence


Open DNA


Recombine with DNA cut with the
same restriction enzyme


Use ligase to seal the cuts and rejoin
the fragments

Restriction enzymes


Experimental Design


Recombinant DNA


Examples of Products of Genetic
Engineering using microbes


Factor VIII


Erythropoetin


Insulin


Interferon


Epidermal growth factor

Industrial applications


Oil “ eating” microbes


Prince William
Sound


Alaska


Degradation of mercury in the
environment


Clean up of
contaminated sites


Agricultural applications


Frost resistant crops


Insecticide resistant crops


Herbicide resistant crops

Transformation with pGlo


PRE
-
INCUBATION

The recipent
E. coli
cells will be exposed to positively
charged calcium chloride (CaCl2) ions. This treatment is
meant to stress the bacterium in order to render its cell
membrane and cell wall permeable to the donar plasmid. This
process will make the recipient
E. coli
"competent" to uptake
the plasmid.

* INCUBATION

The plasmid (with amp+ gene) is added to a recipient
E. coli
suspension, which will now be called
E. coli
+ because it is the
one which is being transformed. Another
E. coli
suspension
will act as a control, called E. coli
-

because it will not be
exposed to the plasmid; therefore, it will NOT inherit the
gene.

* HEAT SHOCK

The recipient cells plus plasmids and the control cells not
exposed to the plasmids are briefly exposed to 42 degrees C.
This step will maximize the uptake of the plasmid through the
wall and membrane of the cells.

Plasmid Vectors



Ori( origin of
replication)


Polylinker cloning sites


Regulatory region


( lac operon)


Antibiotic resistance
gene(s)


Reporter gene for
protein


color or
fluorescent molecule

pGlo


Ori


Polylinker cloning
region


Amp ( beta lactamase
for resistance)


araC( arabinose operon)


pBad


Green fluorescent
protein
-

reporter


pGlo



pGlo


Gene fusion


Transposition of genes from one
location to another on a chromosome


Also can result in the deletion of a
section of a chromosome


Gene fusion has been used with
Pseudomonas syringae, a bacterium
that grows on plants


Pseudomonas syringae


Produce a protein that forms a nucleus
for ice crystals


Ice crystals damage leaves and stems


Removing the gene, prevents damage
to crops when the crops are sprayed
with the resistant forms

Crop damage due to frost


P syringae

on surface of leaves


Protoplast fusion


Removal of the cell wall of organisms
of two strains can result in the
recombination of their genetic
material.


Can select for desirable features of
both strains


Effectively used in yeast, molds, and
plants


Protoplast fusion


Nocardia lactamdurans


produces the
antibiotic cephalomycin


New strains increase the yield of this
important antibiotic

Gene amplification


Bacteriophages or plasmids are
introduced into cells to repliicate or
reproduce at rapid rates


This is used particularly in strains of
bacteria that produce antibiotics


Amplification can also be used to
increase the yield of amino acids,
vitamins, and enzymes

Agrobacterium tumefaciens

and
nature’s genetic engineering


Nature of the Microbe


A. tumefaciens

is a Gram
-
negative,
non
-
sporing, motile, rod
-
shaped
bacterium,


Closely related to
Rhizobium

which
forms nitrogen
-
fixing nodules on
clover and other leguminous plants.


Possesses a large, natural plasmid
called Ti

Agrobacterium tumefaciens


Attracted to wounds or openings in the
plant cell wall


Uses acetosyringone to inject into the plant
cells


Ti plasmid enters the plant cell and
integrates randomly into the host


Plasmid codes or opines and nopalines two
distinctive gene products that lead to tumor
production in infected plants

Ti plasmid


Ti plasmid and genes


ori
--
replication controlled sites



tra region
--
responsible for mobility
from bacteria to plant cell



vir
--
induce uncontrolled cell division
in the host plant



t region (tDNA)
--
group of genes
that control the transfer of the
tDNA to the host chromosome



Genetic Engineering and Ti