南开大学 基因操作原理 第五章

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(1） deletion and addition of restriction sites
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(2） adding genetic markers（LacZ, Amp etc.）
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入gt11 vector
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Application of Insertion vector
blue
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6)Packaging in vitro
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2. Cosmid and Phasmid Vector
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1)Cosmid Cosmid is plasmid with lambda phage cos site. It can clone large foreign DNA fragment (Cosmid itself is smaller), It can infect host by in vitro packaging as λphage, and amplify as plasmid in host. (λcan carry 20kb DNA, whereas cosmid can carry 32-47kb foreign DNA )
cro, product: binding to OL,OR, prevents cI and cro transcripted from PL and PR. cI , the only gene expressing in lysogeny. repressor gene; its product is the inhibitor of early transcription, inhibit cro, keep lysogeny；
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5) Construction of λvector：
(1）deletion and addition of restriction sites； (2）adding genetic markers（LacZ, Amp etc.) (3）screen for Spi- recombinant； (4）introducing Chi site.
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2) Life cycle of Lambda phage
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Red Gam
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3)Expression and control of genes
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Major functions of right operon region:
On the right of N gene concerned with gene regulation and prophage immunity to superinfection. S,R : cell lysis; O,P: DNA replicatios; N,Q : Antiterminator
Chapter 5 Phage and Cosmid vectors
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1. Lambda phage vectors; 2. Cosmid and Phasmid vectors; 3. ssDNA phage vectors; 4. P1 phage vectors.
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mbda phage Vector
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Major functions of central region gene
gam xis att
b2
red （exo and bet）: recombination related, the product is exonuclease , bet ：switch of transition from θ to rolling circle mode gam: encoding E.coli exonuclease V inhibitor, which can inhibit exonuclease V encoded by recB and recC (depredated concatemeric DNA), stead rolling circle mode. int,att,xis relate to integration and prophage excision b2 no essential region
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left operon rigion
A-J: code for head and tail proteins of the phage particle.
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L 48502bp; 2) A circular structure when it is injected in to its host cell； 3）66 genes，3 functional zones; left operon： capsid protein synthesis ； right operon：immunity，gene regulation, DNA synthesis，cell lysis; central operon：recombination，integration
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Characteristics of Cosmid
(1）cloning of large pieces of DNA（eukaryotic gene library construction) (2）only recombinant can be packaged （the size of self ligation is too small to be packaged） (3）to avoid vectors forming multimers, alkaline phosphatase treatment or blunting the end can be used to lower the ligation efficiency
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2) Phasmid
Phasmid is plasmid with λatt site and some other phage genes.
(1) It can integrate intoλphage genome through site-
specific recombination;
1) Structure of Lambda phage genome 2) Life cycle of Lambda phage 3) Expression and control of genes 4) Capacity of λvector 5) Construction of λvector 6) Packaging in vitro
write
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Application of replacement vector
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EMBL3,4
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(3） Spi+ (Sensibility to P2 inhibition)
Wild-type λcan’t grow on E.coli strains of lysogeny for phage P2; in other words, the WTλphage is Spi+ (Sensitive to P2 inhibition)
(2) The recombinant can integrate into host chromosome and become lysogen; (3) Thus foreign gene can first be cloned into plasmid then “lift” into phage, and enhance its infinite
cII: encoding a regulatory protein ,it is necessary for the promoters of int,cI utilize RNA , induce lysogeny. cIII: product: active cI and int, induce lysogeny.
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recombinant（Spi-) E.coli recB,C
activate
Δλred gam
exonuclease V
Chi
promot DNA （ θcircles ）recombination,
produce multimers, because Chi can activate exonuclease V and produce bigger plaques
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1)Structure of Lambda phage genome
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L
R
1）dsDNA, linear, 48502bp; 2) A circular structure when it is injected in to its host cell； 3）66 genes，3 functional zones; left operon： capsid protein synthesis ； right operon：immunity，gene regulation, DNA synthesis，cell lysis; central operon：recombination，integration
bigger plaque.
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WTλ E.coli recB,C
λgam
exonucleaseV
inhibit
exoV inhibitor
promot DNA(θcircles）recombination, produce multimers, which can package with very low efficiency. gam gene product inhibit exonuclease V activity.
shelf-life, and be screened easily (it is usually easier