Download E. coli plasmids

前言
在這一章裡，我們將
Look more closely at the
cloning vector itself
對分子生物學家而言，有 些cloning
vector可用；以及了解它們每一種個別
的特性與用法
以E. coli作為宿主的cloning vectors存在最多種變化
Greatest variety of cloning
vectors exist for use with E.
coli as the host organism
因為E. coli扮演的角色相當重要，因此
過去50年它被研究的相當徹底 ，包括
微生物學 ，生化學，以及遺傳學的研究
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6 Cloning vectors for
E. coli
• most important types of E. coli
cloning vector
• specific uses of representative
molecules
6 Cloning vectors for E. coli
6.1 Cloning vectors based on E. coli plasmids
6.2 Cloning vectors based on M13
bacteriophage
6.3 Cloning vectors based on λ
bacteriophage
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6.1 Cloning vectors based
on E. coli plasmids
Simplest cloning vectors, & ones in
most widespread use in gene cloning,
are those based on small bacterial
plasmids
最小以及用的最廣泛的cloning vector是來自細菌的質體
6.1 Cloning vectors based on
E. coli plasmids
很多的質體vector是可以從
廠商那裡購得，他們把好用
• Many obtainable for的特性全引進質體中了。
commercial suppliers
– Ease of purification所以使用這些質體vector進
行gene cloning就如同家常
– High transformation便飯一般
efficiencyroutine
– Convenient selectable markers
– Ability to clone larger pieces of DNA
• Up to about 8 kb
• One of the first vectors: pBR322
– Still one of the most popular today
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6.1.1 The nomenclature of
plasmid cloning vectors
•
'p' :
– this is indeed a plasmid
•
'BR' :
– identifies laboratory
– Two researchers: Bolivar and Rodriguez
•
'322' :
– distinguishes this plasmid from others
– Also pBR325, pBR327, pBR328
6.1.2 The useful properties of
pBR322
三個重要優點
• Size
 add
– 4363 bp
– Be purified with ease
6kb
• Two sets of antibiotic
resistance genes
– ampicillin or tetracycline
resistance
– used as Selectable markers
• High copy number
– 平時15
– 額外加protein inhibitor
如chloramphenicol可提
高至1000-3000
 A good yield
of recombinant pBR322
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6.1.3 The pedigree
of pBR322
Manipulations
involved in
construction of
pBR322
A summary of the origins of
pBR322
• ampR gene originally
resided on the plasmid
Rl
• tetR gene is derived
from R6-5
• replication origin is
originally from pMB1
– Closely related to
ColE1
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Structure of pBR322 showing the
unique cleavage sites
Plasmid pBR322 is the
most widely used
cloning vehicle
Developed in late 1970s
Paper 1977
Figure 15-9
Detection of
transformed
cells by
insertional
inactivation
Ampicillin (Ap)
Tetracycline (Tc)
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6.1.4 Other typical E. coli
plasmid cloning vector
• pBR327 - a higher copy number plasmid
(non-conjugative)
• pUC8 - a Lac selection plasmid
• pGEM3Z - in vitro transcription of cloned
DNA
pBR327 - a higher copy number plasmid
• Remove 1089 bp
from pBR322
– Left ampR & tetR
gene intact
– Change the
replicative &
conjugative
abilities
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pBR327 & pBR322 有二個很不一樣的地方
pBR327 - a higher copy number plasmid
1. A higher copy number than pBR322
– About 30-45 molecules per E.coli cells
• 特殊情況可達1000
– More suitable
• The aim of the experiment is to study the function of
the cloned gene
2. A non-conjugative plasmid
– Averting biological containment
• 避免A recombinant escaping from test tube
pUC8 - a Lac selection plasmid
• From pBR322: remain replication origin & ampR
gene
• Cloning sites: lacZ'gene - β galactosidase
– Enzyme inactivation
其實此lacZ 基因只
會做出一半的酵素
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三個重要優點
pUC8 has three important advantages
1.
Copy number: 500-700 even before
amplification
–
2.
Identification: a single-step process
–
–
3.
Fortuitous: a chance mutation
plating on to agar medium containing ampicillin plus
X-gal (5-bromo-4-chloro-3-indoly-β-D-galactoside)
Half time
Clustering of the restriction sites
–
–
allows a DNA fragment with two different sticky
ends
Directional cloning
lacZ: β-galasidase
Figure 15-7 Directional cloning
into a plasmid vector (pUC19)
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DNA ligase
X-Gal: 5-bromo-4-chloro-3-indolylβ-D-Galactopyranoside
Figure 15-7 Directional
cloning into a plasmid vector
(pUC19)
agar medium containing ampicillin plus X-gal
α-complementation (figure 15-7)
•
α peptide
– Vectors (pUC19) carry a segment of regulatory
sequences & coding information for first 146 amino
acids of the lacZ gene (β-galasidase)
• Host E. coli cells
– code for carboxy-terminal portion of β-galasidase
• Neither the host-encoded nor the plasmid-encoded
fragment is active, but together they associate to
form an active enzyme
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The pUC plasmids
• The clustering of the restriction sites
– Allows a DNA fragment with two different sticky ends
– Other pUC vectors carry different combinations of
restriction sites
The pUC plasmids
• The clustering of the restriction sites
– Same clusters in equivalent M13mp series
vectors
– Cloned DNA can be transferred directly to its
M13mp counterpart
– Be analyzed by DNA sequencing or in vitro
mutagenesis
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Cloned DNA can be transferred
directly to its M13mp counterpart
The multiple cloning site (MCS) is
inserted into the lacZ gene but does not
interfere with gene function
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pGEM3Z - in vitro transcription of cloned DNA
• Similar to a pUC
vector
專門作RNA的vector，
gene必須放在promoter
後面才能被RNA
polymerase辨認執行
– ampR and lacZ'
genes
• Two promoter for
– RNA polymerase of
T7 bacteriophage
– RNA polymerase of
SP6 phage
Synthesize 1-2 µg of RNA per minute
pGEM3Z - in vitro transcription of cloned DNA
R = RcoRI, SacI,
KpnI, AvaI, SmaI,
BamHI, XbaI,
AccI, HincII, PstI,
SphI & HindIII
Restriction enzyme digest
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6.2 Cloning vectors
based on M13
bacteriophage
6.2 Cloning vectors based on
M13 bacteriophage
• M13 genome is 6.4 kb
– Most: ten closely
packed genes
– Each essential for the
phage replication
• Insert new DNA
– Only a single
intergenic sequence
(507 nts)
– Include the replication
origin
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6.2.1 Development of the cloning
vector M13mp2
• M13mp1
– Introduce the lacZ' gene into the intergenic
sequence
– Forms blue plaques on X-gal agar
6.2.1 Development of the cloning
vector M13mp2
• M13mp2
– GGATTC => GAATTC (EcoRI site)
– aspartic acid => asparagine
– β-galactosidase still perfectly functional
• M13mp2 is simplest M13 cloning vector
– Recombinants are clear plaques on X-gal agar
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6.2.1 Development of the cloning
vector M13mp2
In vitro mutagenesis of the lac region of
M13mp1 to produce M13mp2
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6.2.2 M13mp7 - symmetrical
cloning sites
• Introduce additional restriction sites into the lacZ'
gene
–
–
–
–
Polylinker
EcoRI, BamHI, SalI and PstI
Not totally disrupt the lacZ' gene
Altered β-galactosidase is still produced
• Selection
– Only recombinant M13mp7 phage give clear plaques
• Advantage of M13mp7
– Inserted DNA excised easily using EcoRI
Figure 6.8 Construction of M13mp7
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The derivation of M13mp7
Only recombinant phage give clear plaques
Selection
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Figure 6.10 Recovery of
cloned DNA from a
recombinant M13mp7
P114
A big advantage of M13mp7 is that DNA inserted can
be exised from the recombinant using EcoRI
6.2.3 More complex M13 vectors
• Latest M13 vectors: more complex
polylinkers
• M13mp8 is the counterpart of the plasmid
pUC8
– Take DNA fragments with two different sticky
ends.
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M13mp9 has the same polylinker
but in the reverse orientation
M13mp9 has the same polylinker
but in the reverse orientation
• Important in DNA sequencing
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M13mp9 has the same polylinker
but in the reverse orientation
Important in DNA sequencing
Only ~600 NTs can be read
6.2.3 More complex M13 vectors
• M13mpl0/11 and M13mpl8/19
– Similar to M13mp8/9
– Different polylinkers and different restriction sites
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6.2.4 Hybrid plasmid-M13
vectors: Phagemids
Cloned DNA with M13 vector
–generally 1500 bp, occasionally 3 kb
• Phagemids
– M13 genome with
plasmid DNA
• pEMBL8
– Transfer into pUC8 a
1300 bp fragment of
M13 genome
Structure of pEMBL8(+) &
pEMBL8(-)
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6.2.4 Hybrid plasmid-M13
vectors - pEMBL8
• Signal sequence
recognized by the
enzymes
– DS M13 => SS DNA
• Helper phage
– Provide necessary
replicative enzymes &
phage coat proteins
• Up to 的形式感染細菌
10 kb cloned
以phage
DNA fragments
進去以後以質體的方式存在；
更重要的是改造之後使其容量
提高倒近10 kb
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