Biotechnology and Genetic Engineering-PBIO 450/550

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

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http://bcs.whfreeman.com/lodish5e/pages/bcs
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•
MCB Chapter 12
-
mRNA splicing animation

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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

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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

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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