Transcript Inquiry into Life Twelfth Edition

Molecular Biology
Lecture 4
Chapter 4
Molecular Cloning
Methods
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pUC and b-galactosidase
pUC plasmid
4-2
pUC multiple cloning site
4-3
pUC and b-galactosidase
pUC plasmid
lac promoter
O
MCS
lacZ (a-peptide)
-Induced by lactose or IPTG
-Under the control of the lac repressor
-The MCS is a cluster of sequences recognized by restriction
endonucleases
4-4
pUC and b-galactosidase
a-complementation
Plasmid contains part of the lacZ gene coding for the Nterminal extremity of the b-galactosidase enzyme.
When expressed in E. coli lacZ strain = no activity
Host bacterial strain contains a truncated lacZ gene
encoding a polypeptide missing the N-terminal extremity
When expressed in E. coli = no activity
To use the pUC plasmid for selection, the host strain used
for transformation must be lacZ- and lacZ M15
4-5
pUC and b-galactosidase
a-complementation
The two partial gene products can cooperate to form an
active enzyme
Reconstituted
tetramer
E. coli
lacZ M15
pUC
a-peptide
+
inactive
inactive
Active tetramer
4-6
pUC selection procedure
Plate transformants on a petri dish containing IPTG
(synthetic inducer of the lac operon) and X-gal (synthetic
substrate of b-galactosidase)
a-peptide is synthesized
and complementation
will take place
Blue colony
lacZ gene is disrupted
a-peptide is not synthesized
and complementation
will not take place
white colony
4-7
Non-directional cloning
EcoR1
EcoR1
1)
2)
3)
4)
5)
EcoR1
Cut plasmid DNA and insert with EcoR1
De-phosphorylate the cut vector (alkaline phosphatase)
Mix de-phosphorylated vector and insert + DNA ligase and ATP
Transformation
Selection
De-phosphorylation of cut vector will reduce the number of false positive
4-8
Directional cloning
EcoR1
1)
2)
3)
4)
BamH1
EcoR1
BamH1
Cut plasmid DNA and insert with EcoR1 and BamH1
Mix cut vector and insert + DNA ligase and ATP
Transformation
Selection
Digestion of the vector with two restriction endonucleases prevents
religation
4-9
Summary
4-10
cDNA Cloning
• cDNA is the abbreviation for
complementary DNA or copy DNA
• A cDNA library is a set of clones
representing as many as possible of the
mRNAs in a given cell type at a given time
– Such a library can contain tens of thousands
of different clones
4-11
Making a cDNA Library
4-12
Making a cDNA Library
4-13
Making a cDNA Library
4-14
Making a cDNA Library
4-15
Making a cDNA Library
cDNA
plasmid
Recombinant
plasmid
4-16
Methods of Expressing Cloned
Genes
Cloning a gene permits
• Production of large quantities of a
particular DNA sequence for detailed study
• Large quantities of the gene’s product
(protein or RNA) can also be obtained for
further use
4-17
Expression Vectors
• Vectors discussed so far are used to first
put a foreign DNA into a bacterium to
replicate and screen
• Expression vectors are those that can
yield protein products of the cloned genes
4-18
Fusion Proteins
pUC plasmid
• If inserted DNA is in the
same reading frame as
interrupted gene, a
fusion protein results
– These have a partial bgalactosidase sequence
at amino end
– Inserted cDNA protein
sequence at carboxyl end
4-19
Oligohistidine Expression Vector
• Oligohistidine expression
vector has a short sequence
just upstream of MCS
encoding 6 His
– Oligohistidine has a high
affinity for divalent metal
ions like Ni2+
– Permits purification by
nickel affinity
chromatography
– His tag can be removed
using enzyme enterokinase
without damage to the
protein product
4-20
Summary
• Expression vectors frequently produce
fusion proteins
– One part of the protein comes from coding
sequences in the vector
– Other part from sequences in the cloned gene
4-21
Inducible Expression Vectors
• Main function of expression vector is to yield the
product of a gene – usually more is better
• For this reason, expression vectors have very
strong promoters
• Prefer keep a cloned gene repressed until time
to express
– Large quantities of eukaryotic protein in bacteria are
usually toxic
– Can accumulate to levels that interfere with bacterial
growth
– Expressed protein may form insoluble aggregates,
inclusion bodies
4-22
Controlling the lac Promoter
• lac promoter is somewhat inducible
– Stays off until stimulated
– Actually repression is incomplete or leaky
– Some expression will still occur
4-23
Summary
• Expression vectors are designed to yield
the protein product of a cloned gene
• When a lac inducer is added, cell begins
to transcribe the gene of interest
4-24
Using the Ti Plasmid to Transfer
Genes to Plants
• Genes can be introduced into plants with
specialized vectors
• Common bacterial vector promoters and
replication origins are not recognized by
plant cells
• Plasmids are used containing T-DNA
– T-DNA is derived from a plasmid known as
tumor-inducing (Ti)
– Ti plasmid comes from bacteria that cause
plant tumors called crown galls
4-25
Ti Plasmid Infection
• Bacterium infects plant, transfers Ti
plasmid to host cells
• T-DNA integrates into the plant DNA
causing abnormal proliferation of plant
cells
• T-DNA genes direct the synthesis of
unusual organic acids, opines which can
serve as an energy source to the infecting
bacteria but are useless to the plant
4-26
Ti Plasmid Transfers Crown Gall
4-27
Use of the T-DNA Plasmid
4-28