Genetic Engineering - OpenWetWare

gooseliverBiotechnology

Oct 22, 2013 (3 years and 8 months ago)

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Genetic Engineering

An Overview

What is it???


Applied techniques of genetics and
biotechnology (“Wet lab procedure”). Much
trial and error.


Involves the “isolation, manipulation and
reintroduction of DNA into cells or model
organisms, usually to express a protein’’.


DNA taken from one organism and inserted
(transformed) into another (transgenic)
organism


Heritable, directed alteration of an
organism. Altering DNA or adding new DNA
allows us to change the characteristics of a
cell or cells.

Why do it?


It aims to introduce new
characteristics or attributes
physiologically or physically, such as
making a crop resistant to a herbicide,
introducing a novel trait, or producing
a new protein or enzyme.


For example…

How does it work?


Detect gene within organism of interest


Isolate and “Splice out” DNA


“Ligate” into “Vector” of choice


Introduce into bacteria


Isolate colonies containing vector with gene
of interest


Grow up bacteria colony to produce multiple
copies of gene which is expressed




Isolating and splicing DNA of
Interest…


Conventionally entire genome
fragmented using “Restiction
Enzymes”


E.g. BamH1


Hindiii



Target Palindromic sequences:


AGGTACCT


TCCATGGA






Can result in “Staggered”/”Sticky” ends


AG GTACCT

TCCATG GA


Can result in “Blunt” ends



AGGT ACCT


TCCA TGGA


Enzymes can create sticky ends from blunt ones



PCR can be used to specifically target
gene of interest


“Enzymatic amplification of specific DNA
fragment using repeated cycles of DNA
denaturation, primer annealing and
Chain extension”


Knowledge of full sequence not required


Can produce large amount of copies from

>minute quantity of target DNA


>Partially damaged DNA

Inserting Gene fragments
into Vector of choice


Many types of Vectors depending on
needs


Bacterial Artificial Chromosomes


Yeast Artificial Chromosomes


Plasmids


Use RE to create complimentary sticky
ends in the vector



Possible outcomes post digestion with
R.E.


Plasmid reforms


Fragmented genomic DNA joins up with
itself


Plasmid and fragmented DNA for a hybrid


Contains gene of interest


Contains other gene (unfortunately, most
times!!!)

Insert into Bacteria/Host


Allows expression of protein product


Allows multiple copies of the gene to
be made


Several methods


Heat shock


Electric Pulse (electroporation)


Microinjection/microprojectiles


Viruses


Lysosomes


Identifying the right
colony


3 levels of identification:


Bacteria which have taken up a plasmid


Bacteria which have taken up a
recombinant
plasmid


Bacteria containing the wanted gene


Achieved by using antibiotic resistence
genes and gene probes.

Finally…


Once the correct colony is identified it
is cultured and gene expression
encouraged.


Protein product can be harvested!


However… it is not always successful…
Much patience is required!!!

Some examples of current
applications of genetic
engineering


Synthesis of insulin. Not always
effective due to a eukaryotic protein
being expressed in a prokaryotic cell.


Introduction of resistance genes in
crop plants


Flavr Savr Tomato


Gene therapy (e.g. Cystic Fibrosis)