chapter2Vehicles

1、Chapter 2 Vectors for Gene Cloning: plasmids and bacteriophagesXinjun Yu, 于欣君2015.3.17Contents 2.1 plasmids 2.1.1 Basic features of plasmids 2.1.2 Size and copy number 2.1.3 Conjugation and compatibility 2.1.4. Plasmid classification 2.1.5 Plasmids in organisms other than bacteria The requirements f

2、or a DNA molecule as a cloning vectors Self-replicationSelf-replication： ori siteori site Special phenotypic traitsSpecial phenotypic traits：genetic genetic marker genemarker gene Insert siteInsert site：Multi-cloning siteMulti-cloning site（MCSMCS） Plasmid sizePlasmid size： as small as possibleas sma

3、ll as possible Not affect host cellNot affect host cell2.1.1 Basic features of plasmidsFig.1 Plasmids: independent genetic elements found in bacterial cellsPlasmid is an autonomous self-replicating extra-chromosomal circular DNA特性：1.extra-chromosomal covalently closed circular DNA, cccDNA, size 1-25

4、0kbp. 2.Self-replication autonomous replicon. 3. not essential for host. 4. uncompatibility 5. transformation abilityConformations Plasmids are circular molecules of DNA that lead an independent existence in the bacterial cell. Double-stranded circular DNA covalently closed circular DNA, cccDNAThree

5、 conformations(構(gòu)象) Supercoiled DNA (sc DNA) Open circular (oc DNA) Linear DNA ( L DNA)Genetic marker geneGenetic marker gene Plasmids almost always carry one or more genes, and often these genes are responsible for a useful characteristic displayed by the host bacterium.Some phenotypic traits exhibi

6、ted by plasmid-carried genesAntibiotic resistance抗生素抗性Antibiotic production生產(chǎn)抗生素Degradation of aromatic compounds降解芳香族化合物Haemolysin production生產(chǎn)溶血素Sugar fermentation糖發(fā)酵Enterotoxin production生產(chǎn)腸毒素Heavy-metal resistance重金屬抗性Bacteriocin production生產(chǎn)細(xì)菌素Induction of plant tumours誘導(dǎo)植物瘤Hydrogen sulphide pr

7、oduction生產(chǎn)硫化氫Host-controlled restriction and modification宿主控制的限制與修飾Selectable marker（選擇標(biāo)記）a gene carried by a vector and conferring a Recognizable characteristic on a cell containing the vector or a recombinant DNA molecule derived from the vector.Self-replication: Ori siteSelf-replication: Ori site

8、All plasmids possess at least one DNA sequence that can act as an origin of replication(復(fù)制起點(diǎn)）, so they are able to multiply within the cell quite independently of the main bacterial chromosomeUse replication enzyme of host.Two kinds of plasmidsTwo kinds of plasmids Integrative plasmids (episomes) 整合

9、型質(zhì)粒/游離型質(zhì)粒 Episome is a plasmid able to integrate into the host cells chromosome. Non-integrative plasmids 非整合型質(zhì)粒Size and copy numberThe size of plasmids range from about 1.0kb to over 250kbThe copy number refers to the number of molecules of an individual plasmid that are normally found in a single

10、bacterial cell.Conjugation Plasmids fall into two groups: conjugative and non-conjugative Conjugation: physical contact between two bacteria, usually associated with transfer of DNA from one cell to the other. Conjugative plasmids are characterized by the ability to promote sexual conjugation betwee

11、n bacterial cells, a process that can result in a conjugative plasmid spreading from one cell to all the other cells in a bacterial culture.Compatibility Compatibility (相容性): the ability of two different types of plasmid to coexist in the same cell. Plasmid incompatiblity is the inability of two dif

12、ferent plasmids to coexist in the same cell in the absence of selection pressure. Incompatibility groups(不相容群) comprises a number of different types of plasmid, often related to each other, that are unable to coexist in the same cell.ClassificationCharacteristic coded by the plasimid genesFertility

13、or Fplasmids (F 質(zhì)粒）: carry only tra genes and have the ability to promote conjugal transfer of plasmids.R plasmids (R 質(zhì)粒）: carry genes conferring on the host bacterium resistance to one or more antibacterial agents.Col plasmids (Col 質(zhì)粒): code for colicins, proteins that kill other bacteria, e.g. Col

14、E1.Degradative plasmids (降解質(zhì)粒）: allow the host bacterium to metabolize unusual molecules.Virulence plasmids（毒性質(zhì)粒）: confer pathogenicity on the host bacterium, e.g. Ti plasmids.Classification 1. Depend on whether they carry the tra genes Conjugative( 接合型) : with tra gene non-conjugative (非接合型)plasmid

15、s: no tra genes 2. Based on the copies they carrying : Relaxed plasmids (松弛型): multiple copies per cell stringent (嚴(yán)密型)plasmids: low copies per cell 3. Conjugative plasmids are of relatively high molecular weight and with one to three copies per chromosome, whereas non-conjugative plasmids are of lo

16、w MW and with multiple copies per chromosome.Plasmids in organisms other than bacteria 2m circule (2m 質(zhì)粒): a plasmid found in the yeast Saccharomyces cerevisiae and used as the basis for a series of cloning vectors.pINA13125296 bps10002000300040005000ClaIMluIEco72IBamHIAcc65IKpnISgrAIEcoRIMunISexAIN

17、otIBstBIBssHIITth111IFspIBglIIBstAPIAlw44INotIXbaIHincIISalIStuIBsrGIPshAIEco47IIIBstXIBsu36IScaIAatIIEcoNIAflIIEcl136IISacIUAS1BUAS1BUAS1BUAS1Bmin pLEU2term XPR2zetakanRzetaURA3d119Bacteriophages（噬菌體）Bacteiophages or phages are viruses that specifically infect bacteria.Simply in structure, consisti

18、ng merely of a DNA (or occasionally RNA) molecule carrying genes for replication of the phage, and the capsid made up of protein molecules.20 Two main types of phage structures: a. head and tail 頭-尾(e.g. ) b. filamentous 絲狀結(jié)構(gòu)(e.g.M13)Basic features of bacteriophages21 Three-steps for phage infection

19、 :(1)attach/ inject(2) DNA replicate(3)Capsid components synthesized/ assembled/ released Basic features of bacteriophages22 Lytic infection cycle (溶菌或裂解感染周期溶菌或裂解感染周期): the pattern of infection displayed by a phage that replicates and lyses the host cell immediately after the initial infection. very

20、 short infection cycle ,20min. e.g.T phages Lysogenic infection cycle(溶原感染周期溶原感染周期): the pattern of phage infection that involves integration of the phage DNA into the host chromosome.Two types of infection cycles23Figure 11.1 Lytic development involves the reproduction of phage particles with destr

21、uction of the host bacterium, but lysogenic existence allows the phage genome to be carried as part of the bacterial genetic information. 24Gene organization in the DNA molecule DNA molecule: 49 kb A feature of the genetic map: genes related in function are cluster together.25The linear and circular

22、 forms of The conformation of molecule: linear with two free ends. The linear molecule consists of two complementary strands of DNA. But at either end of the molecule is a single-stranded DNA, a short 12-nucleotide stretch.26Figure2.10 The linear and circular forms of DNA (a) The linear form showing

23、 the left and right cohesive ends. (b) Base-pairing between the cohesive ends results in the circular form of the molecule. (c) Rolling circle replication produces a catenane of new linear DNA molecules, which are individually packaged into phage heads as new particles are assembled. 27The linear an

24、d circular forms of DNA (線形與環(huán)狀 DNA ) Sticky ends (粘性末端）: the ends of a double-stranded DNA molecule where there are complementary single-stranded extensions. Cos site (cos 位點(diǎn)): one of the cohesive, single-stranded extensions present at the ends of the DNA molecules of certain strains of phage. Two r

25、oles of cos sites (cos 位點(diǎn)的兩個(gè)作用): (1) allow the injected linear DNA molecule to be circularized;(2) act as recognition sequences for an endonuclease to cleave at the cos sites, producing individual genomes.28M13- a filamentous phage M13 : filamentous phage M13 DNA molecule :6407 bp, usually in double

26、 stranded circular DNA, sometime showing single-stranded DNA. M13 capsid requiring only three proteins; capsid needing more than 15 proteins. When infection host,M13 does not need genes for insertion its DNA into the host genome.29The infection cycle of M13 M13: lysogenic phage M13 DNA is not integr

27、ated into the bacterial genome New phages are assembled and released from the infected cell continuously, But the cell lysis didnt happened .30 M13- a filamentous phageFig.2.11 The M13 infection cycle, showing the different types of DNA replication that occur.Pilus: one of the structures present on the surface of a bacterium containing a conjugative plasmid, through which DNA is assumed to pass during conjugation.31The attraction of M13 as a cloning vehicle M13作為克隆載體的吸引力 Features of M13 meet the requirements as a cloning vehicle: s