1. 유전자 운반체 (Cloning Vector)

2. Cloning Vector Cloning vector (유전자 운반체)
특정 유전자나 DNA 단편을 자신에 삽입시켜 재조합 DNA를 만든 후
이를 숙주세포에 도입하여
많은 수로 복제될 수 있도록 하거나 (cloning vector)
단백질로 발현될 수 있도록 함 (expression vector)
Cloning vector
Expression vector (발현 운반체)
Shuttle vector
Plasmid vector : pBR322, pUC19, …
Phage vector : λ , M13
Cosmid vector : cosmid (λ), fosmid (F-plasmid), phagemid
Artificial chromosomes : BAC, YAC

3. Features of cloning vectors
Origin of replication (ori, 복제원점)
propagation & maintenance in host
shuttle vector –
Cloning site
restriction sites (제한부위)
multiple cloning site (MCS)
Selectable markers (선발표지)
Antibiotic resistance gene
Auxotrophic selection marker
Reporter (Marker) gene :
lacZα fragment (blue-white selection)
green fluorescent protein (GFP)
luciferase

4. Features of cloning vectors
Elements for expression : expression vector
Promoter : lac promoter
Ribosomal binding site (RBS)
Enhancer
Terminator
Insert size
Copy number
cloning vector : high copy number
expression vector : high copy number ?
Small size
Easy preparation

5. Cloning Vector Vectors for bacteria Vectors for yeast
Plasmid vectors : pBR322, pUC18, …
Phage vector : λ , M13
Cosmid vector : cosmid (λ), fosmid (F-plasmid), phagemid
Bacterial Artificial Chromosome (BAC)
Vectors for yeast
Plasmid vector : shuttle vectors
YEP (yeast episome plasmid), YIP, YRP, YCP
Yeast Artificial Chromosome (YAC)
Vectors for animal
Retrovirus : ssRNA
Adenovirus : dsDNA
Vectors for plant
Ti plasmid (Agrobacterium tumefaciens)
TMV (Tobacco mosaic virus), PVX (Potato virus X)

6. Plasmid Vectors for Bacterial Host
Size : Large
Small
Copy number : Low copy number
High copy number
Conjugation : Conjugative
Non-conjugative
F (fertility) plasmid :
R (resistance) plasmid :
Col (colicin) plasmid :
Degradative plasmid :
Virulence plasmid :

7. pBR322 pBR322 the first widely used E. coli cloning vector
1977 in the laboratory of Herbert Boyer at UCSF
p, plasmid; BR, "Bolivar" & "Rodriguez.“
4361 bp dsDNA plasmid
rep : origin of replication (replicon) : from pMB1
rop : control copy number
can be amplified by chloramphenicol
ampR gene (bla) : β-lactamase (from plasmid RSF2124)
tetR gene : (from pSC101)
unique restriction sites
11 within the tetR gene
6 restriction sites inside the ampR gene

9. pUC18, pUC19 pUC18, pUC19 . small, high copy number, 2686 bp
can be amplified by chloramphenicol
blue-white selection (with IPTG & X-gal)
origin of replication : rep from pBR322
high copy number : lack of the rop gene
a point mutation in rep of pMB1
bla gene : β-lactamase (AmpR), from pBR322
lacZ’ gene : N-termianl portion of β-galactosidase
complemented by host
synthesis can be induced by IPTG
lacI gene : regulatory gene of lac operon
MCS : within the lacZ’ gene
insertion of foreign DNA : destruction of lacZ ’ gene
PUC18 & 19 : MCS arranged in opposite orientations

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11. .
pUC118, pUC119
pUC118, pUC119
pUC18/19 + origin of replication from M13 phage (IG region)
for single strand production

12. T vector T vectors . For cloning of PCR products (T/A cloning)
Taq polymerase : DNA의 3’ 말단에 dA를 첨가함
PCR products : 3’ 말단에 dA 돌출염기 있음 (dA overhang)
3’ 말단에 dT (dU) 돌출 염기를 가지고 있음 (dT overhang)

13. Lambda phage λ phage (Enterobacteria phage λ, coliphage λ) .
dsDNA : 48.5 kb, linear
Escherichia coli
Temperate phage : 잠재성
Lytic cycle (용균성) : 용균반점 (plaque)
Lysogenic cycle (용원성) : prophage
rexA, rexB : 중복감염 (superinfection) 방지 (immunity)
cI : 용균회로에 필요한 유전자 전사 방지

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15. in vitro packaging of λ phage.
in vitro packaging of λ phage
phage의 증식
1.부착 (Attachment)
2. 침투 (Penetration) : 주입 (injection)
3. 생합성 (Biosynthesis) :
초기 단백질 합성 - 효소
유전물질 합성 : λ phage - dsDNA, concatemer
후기 단백질 합성 : protomer (capsomer)
4. 성숙 (Maturation) : 조립과 포장 (Assembly & Packaging)
Self assembly (자가조립)
5. 방출 (Release)
in vitro packaging of λ phage (생체 외 조립)
Protomers (capsomers) + DNA
단일 균주 조립체계 : cos 부위가 손상된 λ phage
파지 조립에 필요한 모든 단백질이 생산되나 조립되지는 않음
이중 균주 조립체계 : 일부 단백질을 만들지 못하는 두 균주 사용

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20. λ phage vectors 왜? … 무엇이 문제인가? 삽입 벡터 (insertional vector) .
in vitro packaging
Lytic cycle (infection) : amplification (burst size)
dsDNA : linear, circular …
무엇이 문제인가?
Lysogeny : 용원성과 관련된 유전자 제거
Packaging : ∓5% (~ 3 kb), 비필수 부분 제거
Restriction sites : 48.5 kb, site-directed mutagenesis
삽입 벡터 (insertional vector)
치환 벡터 (replacement vector)

21. λ phage vectors 삽입 벡터 (insertional vector) 치환 벡터 (replacement vector).
λ phage vectors
삽입 벡터 (insertional vector)
중앙의 비필수 지역 제거, 좌우 팔 연결 후, 독특한 제한부위 도입
35 ~ 40 kb
~ 10 kb 삽입 가능
cDNA cloning, Expression library construction
λgt10, λgt11, λZap
치환 벡터 (replacement vector)
치환될 수 있는 (비필수부위) 양쪽에 제한 부위 도입
제한효소로 처리 후 DNA 단편 치환 삽입
~ 23 kb 삽입 가능
λFIX, λEMBL3, λEMBL4
λ phage DNA replication :
linear DNA – circular DNA : cos (12 nucleotides)
회전원 복제 (Rolling circle mechanism)
concatemer (연쇄체)

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23. M13 Bacteriophage M13 phage Replication of viral ssDNA .
filamentous bacteriophage (선형, 섬유상)
circular single stranded DNA (ssDNA) : 6407 nucleotides
receptor at the tip of the F pilus of the host E. coli
Infection is not lethal - chronic infection
숙주세포가 분열하는 동안에도 바이러스 방출
decreased cell growth : turbid plaques in E. coli
Replication of viral ssDNA
Viral (+) ssDNA enters cytoplasm
Complementary (-) strand is synthesized : dsDNA
Negative supercoiling of dsDNA :
RF (replicative form) DNA (복제형)
Replication of viral (+) strand DNA
by rolling circle mechanism

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25. M13 phage vectors RF (replicative form, 복제형) M13 phage vectors .
Double-stranded DNA : plasmid vector와 같이 취급
Recombinant M13로 형질감염(transfection)된 대장균
ssDNA를 가진 파지 입자 분비
M13 phage vectors
ssDNA 획득
Site-directed mutagenesis
Sequencing by dideoxy method
Probe (탐침)
insert size : ~ 1,500 bp
Instability :
M13mp18 RF phage vector

26. Cosmid vectors Cosmid Cosmid vector
Hybrid plasmid that contains a λ phage cos sequence
cos site (from λ phage) + plasmid = cosmid
Can replicate as plasmid (origin of replication)
Colony formation on a selection medium
Can be packaged in phage capsids
if 45 kb ~
allows transduction (형질도입)
No lytic cycle : no plaque formation
Cosmid vector
Loading capacity : 40 ~ 45 kb
Genomic library (유전자 도서관)
Unstable, difficult to maintain

27. Cloning with cosmid vector.
Cloning with cosmid vector
1. Restriction digestion of cosmid
2. Insertion of the DNA fragment into the restriction site
3. in vitro packaging of phage with recombinant cosmid
4. Infection of E. coli with phage(cosmid)
5. Growth of E. coli (on selection medium)

28. Fosmid Fosmid . Similar to cosmid :
in vitro packaging into phage particles
Transfection (형질감염)에 의해 숙주세포에 도입
Based on F-plasmid
only one fosmid molecule per host cell
relatively higher stability DNA inserts of up to 40 kb
Fosmid library

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30. Phagemid vector Phagemid (phasmid) .
Plasmid + origin of replication from f1 filamentous phage
f1 ori : for replication of ssDNA, for packaging
also with an origin of replication for dsDNA replication
replicate as a plasmid
packaged as ssDNA in viral particles
used in combination with M13 phage (helper phage)
ssDNA for sequencing purposes.
for site-directed mutagenesis.
to generate hybridization probes for mRNA or cDNA

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32. Bacterial Artificial Chromosome (BAC). .
Bacterial Artificial Chromosome (BAC)
Bacterial Artificial Chromosome (BAC, 세균 인공염색체)
Based on F-plasmid (fertility) :
Conjugation (접합)
Low copy number : 1 ~ 2 plasmids/cell
Partition genes (parA, parB)
세포 분열 중 plasmid가 딸세포로 정확히 나누어 들어가게 함
(even distribution)
재조합 DNA의 재배열, 재조합 방지
Usual insert size : 150 ~ 350 kb
for genome projects
F-plasmid : closed circular dsDNA – easy to treat
For E. coli
* PAC : based on bacterial P1-plasmid.

33. Bacterial Artificial Chromosome (BAC). .
Bacterial Artificial Chromosome (BAC)
Common gene components
repE : for plasmid replication & regulation of copy number
parA & parB : for partitioning F plasmid to daughter cells
for stable maintenance of the BAC
selectable marker for antibiotic resistance
lacZ at the cloning site for blue/white selection
T7 & Sp6 phage promoters for transcription of inserted genes

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pBAC108L

35. Yeast Cloning Vectors Yeast Yeast cloning vectors .
Tough polysaccharide cell wall : barrier to DNA
Removal of cell wall required
Spheroplast can take up DNA following CaCl2 treatment
yeast plasmid discovered : plasmid vector
no yeast virus
Yeast cloning vectors
Plasmid vector : based on 2 μm (2 micron) circle
Yeast episomal plasmid (YEp)
Yeast replicative plasmid (YRp)
Yeast centromeric plasmid (YCp)
Yeast integrative plasmid (YIp)
Yeast Artificial Chromosome (YAC)
* Shuttle vectors : E. coli & Yeast

36. Yeast episomal plasmid (YEp) vector.
Yeast episomal plasmid (YEp) vector
Episome :
숙주세포 내에서 독립적으로 증식할 수 있을 뿐 아니라
숙주세포의 염색체에 삽입되어 숙주 염색체와 함께 복제됨
F-plasmid (Hfr), λ phage
2 μm plasmid
Native yeast plasmid : Saccharomyces cerevisiae
6.3 kb (6,318 bp)
Maintained stably at high copy number
YEp (Yeast episomal plasmid) vector
based on 2 μm plasmid
origin of replications from 2 μm plasmid (Yeast)
origin of replication from pBR322 (E. coli)
shuttle vector :

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ori : REP1 & REP2 : even distribution FLP : control copy number D : repress FLP expression

38. YEp vectors . Shuttle vector : S. cerevisiae & E. coli
ori from 2 μm plasmid, ori form pBR322
Selectable markers
from pBR322 : TetR & AmpR
from yeast : LEU2 (an enzyme for leucine biosynthesis)
His3 (an enzyme for histidine biosynthesis)
Ura3 (an enzyme for uracil biosynthesis)
숙주 효모 : 특정 유전자 결손
특정 선택표지 유전자를 포함한 운반체로 형질전환된 효모 균주만이
특정 영양소가 없는 선별배지(최소배지)에서 증식할 수 있음
Multiple cloning site (MCS)
Episome : replicate independently
integrate into yeast chromosome
(homologous recombination of a yeast gene)

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40. Yeast centromeric plasmid (YCp).
Yeast centromeric plasmid (YCp)
Centromere : 동원체
염색체처럼 세포 분열시마다 1회만 복제
mini-chromosome vector
Yeast integrative plasmid (YIp)
bacterial plasmid + yeast gene (for selection)
Nothing from 2 μm plasmid : no ori for yeast
Must be integrated into yeast genome to be maintained
One copy per cell
YRp (Yeast replicative plasmid) vector
ARS (autonomously replicating sequence) vector
ARS from a yeast chromosome

41. Yeast Artificial Chromosome (YAC).
Yeast Artificial Chromosome (YAC)
효모 염색체처럼 기능하는 선형 운반체
Bacterial plasmid + yeast genes
거대 유전자의 발현과 기능 분석 (~ 1,000 kb, 단백질 번역 후 수식)
pYAC3
ARS sequence for replication in yeast (복제원점)
CEN4 sequence for centromeric function (동원체)
Telomeric sequences at the two ends (말단체)
Protection from exonuclease action
Selectable markers from yeast
영양소 합성 유전자 (Trp1, Ura3 )
Selectable marker for DNA insertion (SUP4)
with inserted DNA : red colongy
without inserted DNA : white colony
Sequences from plasmid : selection & propagation in E. coli

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43. Vectors for Plant & Animal.
Vectors for Plant & Animal
Plant
Ti plasmid (Agrobacterium tumefaciens)
Viral vector
DNA virus : Cauliflower mosaic virus (CaMV)
RNA virus : Tobacco mosaic virus (TMV)
Particle Bombardment : 유전자 총
Animal
Vector : Virus + Plasmid +
Electroporation
Microinjection
Liposome