Transcript Unit 4 Notes for pGlo lab

What is Transformation?
Change…
In molecular biology,
change in which
genetic material
carried by an
individual cell is
altered by
incorporation of
foreign DNA
Objective 1 - Define the term plasmid and explain
its significance for bacteria and recombinant
technology.
• A plasmid is a small piece of circular DNA in
bacteria in addition to bacterial chromosome
• Typically contain genes for traits beneficial for
survival such as antibiotic resistance
• In nature, bacteria transfer these plasmids back
and forth
• Acts as a vector – a gene taxi to get new DNA into
bacteria
Constructing a Plasmid
• Specific fragments of DNA may be
isolated, cut into discrete pieces by
the action of restriction enzymes, and
rejoined by the action of DNA ligase
http://www.dnai.org/b/index.html
Objective 2 - Describe how genomic and plasmid
DNA can be isolated from cells.
How is DNA isolated?
http://www.iupui.edu/~wellsctr/MMIA/is
olating_dna/dna_isolation_rev.swf
Objective 3 - Explain how the creation of sticky ends
by restriction enzymes is useful in producing a
recombinant DNA molecule.
Also called restriction endonucleases
1962: “molecular scissors” discovered
in in bacteria
E. coli bacteria have an enzymatic
immune system that recognizes and
destroys foreign DNA
3,000 enzymes have been identified,
around 200 have unique properties,
many are purified and available
commercially
Restriction Endonucleases
Recognition sites have symmetry
(palindromic)
“Able was I, ere, I saw Elba”
5’-GGATCC-3’
Bam H1 site:
3’-CCTAGG-5’
Restriction Enzymes
Enzymes recognize specific 4-8 bp sequences
Some enzymes cut in a staggered fashion - “sticky
ends”
EcoRI
5’…GAATTC…3’
3’…CTTAAG…5’
Some enzymes cut in a direct fashion – “blunt ends”
PvuII
5’…CAGCTG…3’
3’…GTCGAC…5’
Why don’t bacteria destroy their own DNA
with their restriction enzymes?
Methylation!!
Why are sticky ends better?
• The most useful restriction enzymes cut
DNA in a staggered way, producing
fragments with “sticky ends” that
bond with complementary sticky ends
of other fragments.
• Blunt ends do not have the “overlap”
to bond with.
Sticky end restriction enzymes in
transformation
Human DNA cleaved with EcoRI
5’-C-G-G-T-A-C-T-A-G-OH
3’-G-C-C-A-T-G-A-T-C-T-T-A-A-PO4
Corn DNA cleaved with EcoRI
+
PO4-A-A-T-T-C-A-G-C-T-A-C-G-3’
HO-G-T-C-G-A-T-G-C-5’
Complementary base pairing
5’-A-C-G-G-T-A-C-T-A-G A-A-T-T-C-A-G-C-T-A-C-G-3’
3’-T-G-C-C-A-T-G-A-T-C-T-T-A-A G-T-C-G-A-T-G-C-5’
+ DNA Ligase, + rATP
5’-A-C-G-G-T-A-C-T-A-G-A-A-T-T-C-A-G-C-T-A-C-G-3’
3’-T-G-C-C-A-T-G-A-T-C-T-T-A-A-G-T-C-G-A-T-G-C-5’
recombinant DNA molecule
Restriction enzyme animation
http://www.dnai.org/b/index.html
Go to Techniques – cutting and
pasting
Objective 4 - Explain each of the steps taken to
isolate a gene of interest and insert it into bacterial
DNA to produce the desired protein.
Recombinant
Plasmid
E. coli host cell
Transformed Bacteria
http://www.sumanasinc.com/webcontent/animations/content/plasmidcloning.html
Making bacterial clones
The genomic DNA and a bacterial plasmid are
isolated
Both are digested with the same restriction enzyme
The fragments are mixed, and DNA ligase is added to
bond the fragment sticky ends
The solution with the transformed plasmid is added to
bacteria.
Bacteria take up plasmid
Bacteria are grown in culture dishes, creating “clones”
that should all have the plasmid in them
But how do we check?
Objective 5 - Identify ways to determine if genetic
transformation has been successful.
A clone carrying the gene of interest can be identified
with a nucleic acid probe having a sequence
complementary to the gene
The plasmid may also incorporate an ampicillin
resistance gene, then the bacteria grown in the
presence of ampicillin, only the bacteria with the
plasmid of interest should survive.
A gene for fluorescent protein may be incorporated
into the plasmid with the gene of interest, showing
which organisms now carry the gene of interest
Ampicillin
Resistance Gene
Bioluminescence
Gene
pGreen
Plasmid
Bioluminescent AND antibiotic resistant
Objective 6 - Describe how newly formed protein is
isolated from transformed bacterial cells.
• Wash cells from plates and centrifuge,
discard liquid
• Lyse bacterial cells in a non-denaturing
environment (protease inhibitors and
buffer solution)
• Centrifuge and collect the liquid for
soluble proteins and the pellet for non
soluble protein
• Check with gel electrophoresis
Objective 7 - Relate bacterial transformation to real
life applications used by society.
• Insulin
• Growth hormone
• Interferon
Information from (among others) microvet.arizona.edu/Courses/MIC205/10GenEng/GeneticEng
Ineering and www.udel.edu/chem/bahnson/chem527