Biotechnology and Genetic Engineering
PBIO 450/550
•
Eukaryotic gene organization
•
Restriction enzymes
•
Cloning vectors
Eukaryotic gene organization
enhancers
silencers
Eukaryotic gene organization & RNA processing
Basic Transcriptional Mechanism and
mRNA Splicing Animations
•
MCB Chapter 4
-
Basic Transcriptional Mechanism animation
•
http://bcs.whfreeman.com/lodish5e/pages/bcs
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MCB Chapter 12
-
mRNA splicing animation
•
http://bcs.whfreeman.com/lodish5e/pages/bcs
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Prokaryotic vs. eukaryotic gene organization
Alternative splicing of eukaryotic 1
°
RNA transcripts
Eukaryotic gene expression
MCB Chapter 4
-
Life Cycle of mRNA
•
http://bcs.whfreeman.com/lodish5e/pages/bcs
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Recombinant DNA cloning procedure
Recombinant DNA cloning procedure
•
See MCB Chapter 9
–
Plasmid Cloning
•
http://bcs.whfreeman.com/lodish5e/pages/bcs
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Restriction enzymes & DNA methylation
Recognition sequences of some REs
Enzyme
Recognition site
Type of cut end
Eco
RI
G
↓
A
-
A
-
T
-
T
-
C
5’ P extension
Bam
HI
G
↓
G
-
A
-
T
-
C
-
C
5’ P extension
Pst
I
C
-
T
-
G
-
C
-
A
↓
G
3’ P extension
Sau
3A1
↓
G
-
A
-
T
-
C
5’ P extension
Pvu
II
C
-
A
-
G
↓
C
-
T
-
G
Blunt end
Hpa
I
G
-
T
-
T
↓
A
-
A
-
C
Blunt end
Hae
III
G
-
G
↓
C
-
C
Blunt end
Not
I
G
↓
C
-
G
-
G
-
C
-
C
-
G
-
C
5’ P extension
Mapping of restriction enzyme sites
Vector system
Host cell
Insert capacity (kb)
Plasmid
E. coli
0.1
-
10
Bacteriophage
l
E. coli
10
-
20
Cosmid
E. coli
35
-
45
Bacteriophage P1
E. coli
80
-
100
BAC (bacterial artificial
chromosome)
E. coli
50
-
300
P1 bacteriophage
-
derived AC
E. coli
100
-
300
YAC
Yeast
100
-
2,000
Human AC
Cultured human cells
>2,000
Cloning vectors and their insert capacities
Plasmid cloning vectors
Three important features
1.
Cloning site
2.
Ori
-
an origin of replication
3.
A selectable marker (amp
r
)
pBR322
The plasmid pBR322 is one of the most commonly used
E.coli
cloning vectors. pBR322 is 4361
bp in
length and contains: (
1
) the replicon
rep
responsible for the replication of plasmid (source
–
plasmid
pMB1); (
2
)
rop
gene coding for the Rop protein, which promotes conversion of the unstable RNA
I
–
RNA
II complex to a stable complex and serves to decrease copy number (source
–
plasmid pMB1); (
3
)
bla
gene, coding for beta
-
lactamase that confers resistance to ampicillin (source
–
transposon Tn3); (
4
)
tet
gene, encoding tetracycline resistance protein (source
–
plasmid pSC101).
ori
pUC18/19
pUC18 and pUC19 vectors are small, high copy number,
E.coli
plasmids,
2686
bp in length. They are identical except that they contain multiple
cloning sites (MCS) arranged in opposite orientations. pUC18/19 plasmids
contain: (
1
) the pMB1 replicon
rep
responsible for the replication of
plasmid (source
–
plasmid
pBR322
). The high copy number of pUC
plasmids is a result of the lack of the
rop
gene and a single point mutation
in
rep
of pMB1; (
2
)
bla
gene, coding for beta
-
lactamase that confers
resistance to ampicillin (source
–
plasmid pBR322); (
3
) region of
E.coli
operon
lac
containing CAP protein binding site, promoter Plac,
lac
repressor
binding site and 5’
-
terminal part of the
lacZ
gene encoding the N
-
terminal
fragment of beta
-
galactosidase (source
–
M13mp18/19
). This fragment,
whose synthesis can be induced by
IPTG
, is capable of intra
-
allelic (alfa)
complementation with a defective form of beta
-
galactosidase encoded by
host (mutation
lacZ
DM15). In the presence of IPTG, bacteria synthesize
both fragments of the enzyme and form blue colonies on media with
X
-
Gal
.
Insertion of DNA into the MCS located within the
lacZ
gene (codons 6
-
7 of
lacZ
are replaced by MCS) inactivates the N
-
terminal fragment of beta
-
galactosidase and abolishes alfa
-
complementation. Bacteria carrying
recombinant plasmids therefore give rise to white colonies.
pGEM
-
3Z
Cloning foreign DNA into a plasmid vector
Alkaline phosphatase
-
removes
5’ phosphate (P) groups of DNA
molecules; BAP is more stable
but less active than CIP
T4 DNA ligase
–
joins 5’
phosphate (P) groups of DNA
molecules to 3’ hydroxyl (OH)
groups of DNA
Some antibiotics commonly used as selective agents
Antibiotic
Description
Ampicillin
(Amp)
Inhibits bacterial cell wall synthesis; inactivated by
b
-
lactamase, which cleaves the
b
-
lactam ring of amp
Hygromycin
B (
HygB
)
Kanamycin
(Kan)
Binds to 30S ribosomal subunit and inhibits protein
synthesis; inactivated by a
phosphotransferase
Neomycin (Neo)
Binds to 30S ribosomal subunit and inhibits protein
synthesis; inactivated by a
phosphotransferase
Streptomycin (Str)
Tetracycline (Tet)
Binds to 30S ribosomal subunit and inhibits protein
synthesis;
tet
r
gene encodes a protein which prevents
transport of
tet
into the cell
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