Recombinant Cloning - lucu

neighgreasycornerBiotechnology

Dec 14, 2012 (4 years and 9 months ago)

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www.letstalkscience.ca/challenge.html


Biology:
study of living things, from simple
viruses and single
-
celled organisms to the
most complex ecosystems.




Microbiology:
The study of very small
organisms.




Single
-
celled organism around 3 000 nm in
diameter. (human
rbc

~ 10 000 nm)



The number of bacteria in your
mouth
is larger
than the number of people who have ever lived
on earth.



Earliest fossils of prokaryotes (subsection of
bacteria) are over 3.5 billion years old.



Bacteria have
evolved
into a wide variety of
different types and have adapted to variety of
different environments.



Cocci
: live in humans and animals


does not commonly cause
disease.



Bacilli
: live in lower intestines of
birds and mammals. Some strains
(i.e. E. coli) can cause serious
disease and even death



Spirilla
: Common to poultry, lives
in the digestive track of many
birds. One of the most common
causes of food poisoning.



Vibrios
:
Live in marine
environments (i.e. costal reefs) and
can cause infection after eating
seafood (i.e. undercooked oysters)




Bad bacteria are
pathogenic

(cause diseases)



Examples: tetanus, typhoid fever,
tuberculosis, strep throat, anthrax and food
poisoning.



Some bacteria can even cause
Necrotizing
fasciitis (
flesh eating disease)



Many bacteria can be helpful and promote
good health



i.e.
Probiotic

bacteria ( live in our digestive
system and prevent harmful germs from
growing there)



Bacteria is also used to make certain foods
like vinegar, sauerkraut and yogurt.



Recombinant DNA technology emerged as a
response to the need for specific DNA
segments in amounts sufficient for
biochemical analysis. The method entails
clipping the desired segment out of the
surrounding DNA and copying it millions of
times


Bacteria store of their genes
(pieces of DNA that code
proteins) in a single molecule
of DNA.



But also contain mobile
segments (plasmids) used for
antibiotic resistance.



Theses plasmids aren’t
required for normal growing
conditions, but are very helpful
in aiding the bacteria fight off
antibiotics such as penicillin,
ampicillin
, etc…which would
normally kill the cell.


The plasmids (also known as vectors) are
what scientists use to introduce “genes of
interest” to learn more about the gene’s
function, structure or sequence (the DNA
coding)


The process of introducing genes into a
vector to form a new DNA molecule which can
be replicated in a host cell is called
Recombinant Cloning



With recombinant cloning two different
strands of DNA that would not normally occur
together are combined and multiple copies
are created.



1.
Ligation Reactions:
transferring the “gene
of interest”. Scientist use special proteins
called
restriction enzymes
to cut both the
gene and the plasmid at specific sites that are
complementary to each other.



The two loose pieces of DNA find each other
with the aid of
DNA
ligase

and are joined
(
ligate
) to become a
modified plasmid.


The plasmid cannot clone a gene on its own. It needs
a host system to make copies of the plasmid.



The most efficient host system is bacteria (specifically
E. coli because they divide and grow rapidly)



To make it easier for the bacteria to take in the
plasmids they are grown in a mixture of plasmids and
salts.



Osmotic pressure and the presence of the plasmids
will cause the plasmids to enter the
E.coli

cells




Cells must be screened to determine which
cells picked up the plasmids and which did
not.



Plasmids carry selectable makers, usually a
gene that codes antibiotic resistance.



Since the bacteria are grown in the presence
of an antibiotic only the bacteria with the
plasmids will be able to live


Final step is to harvest the plasmids from the
bacteria.



To do this the bacteria are
lysed

(cell membranes
are broken open) and the plasmids are separated
from the bacteria using an acidic solution which
is high in salt (because plasmid DNA can
withstand these conditions, but regular DNA
cannot)



Finally the plasmids are separated from all other
parts using
centrifugation (
spinning a high
speed)



Bacteria that are able to degrading
xenobiotic

chemicals into
harmless products.



The genus Pseudomonas is capable of detoxifying more than
100 different compounds.



They are able to do this because they carry genes that code for
enzymes which are able to break down toxic compounds.



This process is very slow and not always a perfect process.



Through genetic engineering scientists can combine many
different plasmids (which contain the
degradative

enzymes) and
combine them to a single strain of bacteria and create a “super
-
bug”


Very small (17 ~ 400 nm) 100 times smaller than the
average bacteria.



Made up of 3 basic parts:


1. Nucleic acid : set of genetic material (either DNA or
RNA) packaged in a protein shell



2.
Capsid
: a protein coat that surrounds DNA or RNA to
protect it



3. Envelope: a covering for the
capsid

that is made up of a
mix of proteins, fats and carbohydrates (complex sugars).



* Not all viruses have envelopes. The ones that don’t have
are called naked or non
-
enveloped.


Helical viruses:
resembles
long rods. Example of virus:
influenza



Polyhedral viruses:
many
-
sided. Example of virus:
adenovirus which cause
respiratory illnesses



Enveloped viruses:
spherical
in shape due to the
protein,fat

or
carb

coat.
Example of virus : HIV



Complex viruses:
have
complicated structures
attached. Example of virus:
bacteriophage

which infects
bacteria


Viruses do not have a metabolism.


Not considered alive


Lack the ability to reproduce


Needs a host cell to live and make more viruses.



5 Step process:



1. Attachment : the virus attaches to a host cell



2. Penetration: the nucleic acid of the virus moves through the cell membrane into
the host cell.



3. Replication: virus forces host to produce the necessary components for its
reproduction



4. Assembly: the newly produced virus components are assembled into new
viruses.



5. Release: the complete viruses are released from the cell and infect other cells.


Our immune system is stimulated when it
detects an antigen and creates antibodies.



Antigens are found on the surface of the viral
envelop.



The antibodies (proteins)and specialized cells
destroy the intruder.




Vaccination

is the administration of antigenic
material (a vaccine) to stimulate the immune
system of an individual to develop
adaptive

immunity to a disease. Vaccines can prevent
or ameliorate the effects of infection by many
pathogens.



The virus in the vaccine is either inactivated
or attenuated (alive but not
infectuous
)