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Genetic
Engineering
What is Genetic Engineering?
• Genetic Engineering = inserting a foreign
gene of interest into a host to transcribe and
translate a particular protein.
• Ex. Inserting the human insulin gene into
bacteria to mass produce it.
Image taken without permission from http://www.medicalprogress.org/uploads/images/insulin%20inject%20WSU%20210.jpg
General Steps
• Obtain the gene of interest (ex. insulin
gene)
• Insert the gene into the host (ex. bacteria)
• Allow the host to multiply and express the
foreign gene
– get your desired protein!
– Get lots of cells that can make the protein =
clones
The Big Picture
• The inserted gene is transcribed and
translated using the RNA Polymerase,
ribosomes and other resources in the cell
Plasmids
chromosomal DNA
• Circular DNA
• Extrachromosomal
– NOT part of the E. coli genome
– “extra” DNA
plasmids
• Contain a few non-essential genes
• Can give the bacteria additional “traits”
– Depends on the genes on the plasmid
• Can be exchanged between bacteria
Recombinant plasmids
• Plasmids can be modified in biological labs
• Modified plasmid = Recombinant plasmid
• Plasmids can be used as cloning vectors to
get the recombinant plasmid into E. coli
– Cloning vectors = way to get the gene of
interest into the host
Transformation
• Process in which
foreign DNA is
physically inserted into
host E. coli cells.
• E. coli that contains
recombinant plasmid =
Transformed cell
Image taken with out permission from
http://summerschool.at/static/irismaria.schoenbrunner/imag
es/transformation.png
Transformation Steps
• Recombinant plasmids
and host E. coli are mixed
together
• CaCl2 is added
– The Ca2+ ions neutralize
the negative charges on
plasmid DNA
– Help plasmid enter the
membrane
Image taken without permission from
Transformation Animation. Available at
http://www.dnai.org/b/index.html
Transformation Steps
• Heat Shock
– By rapidly changing the temperature of the
solution, temporary pores are opened in the
membrane
– Creates an opening for the plasmids to enter the
E. coli
Transformation
• Transformation is not 100% successful
• After transformation
– Some cells will contain plasmid = transformed
– Some cells won’t contain plasmid =
untransformed
• In a later step, you will determine which
cells were transformed
E. Coli as a host
• E. coli is a good host because:
– Reproduce quickly (once every 20 minutes)
– Nonpathogenic (the strain we use is not
harmful)
– Genome fully characterized (all genes have
been sequenced)