基因工程基因工程chapter06

1、.Cloning vectors based on E.coli plasmidsCloning vectors based on M13 bacteriophage Cloning vectors based on bacteriophageandotherhighcapacityvectorsenablegenomic libraries to be constructedVectors for other bacteria5.Cloning vectors for E.coli6.1 Cloning vectors based on E.coli plasmids1.The

2、 nomenclature of plasmid cloning vectors质粒载体的系统命名法pBR322Distinguish from other developed vectors in the same lab, such as pBR325, pBR327, pBR328, etc.plasmidBolivar and Rodriguez (researchers initial name or Lab. name etc)2.The useful properties of pBR322 small size two markers (ampR and tetR) ,clon

3、ing sites can produce sticky ends high copy number usually contains 15 ,1000-3000 copies can obtained by plasmid amplificationOrigin of replication3.Other typical E.coli plasmid cloning vectors1089 bp Higher copy number30-45 The conjugative ability was lostAdvantages: higher copy number (500-700) a

4、single selection step MCS and stiky endspUC8a Lac selection plasmidpGEM3Zin vitro transcription of cloned DNAVery similar to pUCTheRNA thatisproducedcouldbeusedasa or for athybridizationprobe, requiredaimedmightbeexperimentsstudyingRNAprocessing(e.g., the removal of introns) or protein synthesis1.2.

5、3.4.Cloning vectors based on E.coli plasmidsCloning vectors based on M13 bacteriophage Cloning vectors based on bacteriophageandotherhighcapacityvectorsenablegenomic libraries to be constructedVectors for other bacteria5.Cloning vectors for E.coli6.2 Cloning vectors based on M13 phageIntergenic Sequ

6、ence507nucleatidesIntroduce the lacZ gene into the intergenic sequenceM13mp7There is a limit to the size of DNA fragment that can be cloned with an M13 vector, with 1500 bp generally being looked on as the maximum capacity, though fragments up to 3 kb have occasionally been cloned.To get around this

7、 problem a number of hybrid vectors (“phagemids”) have been developed by combining a part of the M13 genome with plasmid DNA.1300 bpdouble-strandedM13single- before phageCan insertmolecule stranded secretion particlesinto DNA10kb andDNA,canofnewselection by standardway agaroncontaining X-galPhagemid

8、s(噬粒) Hybrid plasmid-M13 vectorspEMBL8 cloning experiments aresubsequently infected with normal M13 to act as a helper phage, providing the necessary replicative enzymes and phage coat proteins..Cloning vectors based on E.coli plasmidsCloning vectors based on M13 bacteriophage Cloning vectors

9、 based on bacteriophageandotherhighcapacityvectorsenablegenomic libraries to be constructedVectors for other bacteria5.Cloning vectors for E.coli6.3Cloning vectors based on phageWhy develop or use vectors ?Insert size: Plasmids: Phagemids: vectors: 6kb 10kb 25kbNaturalgenomeTwo problems049 kb2035 Se

10、gments of the genome can be deleted without impairing viability2035kb, non-essential region Natural selection can beused to isolate modified that lack certain restriction sitesToo many restriction enzyme recognition sitesin vitro mutagenesis Natural selectionFigure 6.15Using natural selection to iso

11、late phage lacking EcoR Irestriction sites. Two kinds of vectors: Insertion vectors gt10 (see Fig. 6.16) ZAPII (see Fig. 6.16) Replacement vectorsGEM11, GEM12 EMBL4 Insertion vectorsTurbid ClearClear blue and whitegt10can carry up to 8 kb of new DNATurbid ClearZAPIIcan carry up to 10 kb of new DNACl

12、ear white Replacement vectorsReplacement vectors are generally designed to carry larger pieces of DNA than insertion vectors.Recombinant selection is often on the basis of size, with non-recombinant vectors being too small to be packaged into phage heads.EMBL4 can carry up to 20 kb of inserted DNA C

13、osmid （粘粒,柯斯质粒） Plasmid cos siteA cosmid is basically a plasmid that carries a cos site,aselectablemarker,aplasmidoriginofreplication,but lacks all the genes.Long DNA fragment can be cloned using cosmid. Plaques become to coloniespJB8Cloning experiment.Cloning vectors based on E.coli plasmids

14、Cloning vectors based on M13 bacteriophage Cloning vectors based on bacteriophageandotherhighcapacityvectorsenablegenomic libraries to be constructedVectors for other bacteria5.Cloning vectors for E.coli6.4 and other high capacity vectors enablegenomic libraries to be constructedGenomic library：a co

15、llection of clones sufficient in thenumber to include all the genes of a particular organism by using the genomic DNA.cDNA library： a collection of clones sufficient in the number to include all the genes of a particular organism by using the complementary DNAs of mRNAs.Genomic Library:How many clon

16、es are needed for a genomic library.ln(1 - P)N =ln(1 - a/b)Average size of the DNA fragmentsNumber of clones requiredTotal size of genomeProbability(eg.A 95% probability that any given gene is present)Number of clones needed for genomic libraries of a variety of organismsArtificial chromosomes: BACc, PACs, YACs, HACs,bacterial artificial chromosomes (BACs)300 kb.P1-derived artificial chromosomes (PACs).Cloning vectors based o