Plasmids are excellent cloning vectors Figure 5.21 - - PowerPoint PPT Presentation

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Feb 22, 2023 207 likes •437 views

Plasmids are excellent cloning vectors Figure 5.21 Figure 11.11 Order of restriction enzyme cut sites in polylinker Ampicillin lacZ resistance Apo I - Eco RI Ban II - Sac I Acc 651 - Kpn I Ava

SLIDE 1

Plasmids ¡are ¡excellent ¡ ¡ cloning ¡vectors ¡

Figure ¡5.21 ¡

SLIDE 2

Figure 11.11

Ampicillin resistance Polylinker Origin of DNA replication

pUC19

2686 base pairs lacI lacZʹ″ ʹ″

Order of restriction enzyme cut sites in polylinker

ApoI - EcoRI BanII - SacI Acc651 - KpnI AvaI - BsoBI - SmaI - XmaI BamHI XbaI AccI - HincII - SalI BspMI - BfuAI SbfI PstI SphI HindIII

SLIDE 3

Figure 11.12

AmpR lacZʹ″ ʹ″ Vector Foreign DNA Opened vector Recyclized vector without insert Vector plus foreign DNA insert Transformants blue (β-galactosidase active) Transformants white (β-galactosidase inactive)

Digestion with restriction enzyme Join with DNA ligase Transform into Escherichia coli and select on ampicillin plates containing Xgal

SLIDE 4

Figure 11.18

Capsid genes cos att int xis J N QSR cos Replaceable region

Wild-type lambda Charon 4A

(replacement vector)

Charon 16

(insertional vector) β-Gal gene β-Gal gene Another substitution Another substitution

SLIDE 5

Figure 11.19 Replaceable region Infective lambda virion R R R R L L L L cos

Foreign DNA Hybrid DNA

Digestion with restriction enzymes Ligation with foreign DNA Packaging cloned DNA into phage head Phage assembly

SLIDE 6

Figure 11.13

Well-developed genetics Many strains available Best known bacterium Easily transformed Nonpathogenic Naturally secretes proteins Endospore formation simplifies culture Well-developed genetics Nonpathogenic Can process mRNA and proteins Easy to grow Potentially pathogenic Periplasm traps proteins Genetically unstable Genetics less developed than in E. coli Plasmids unstable Will not replicate most bacterial plasmids Advantages Disadvantages Escherichia coli Bacillus subtilis Saccharomyces cerevisiae

Bacteria Eukaryote

SLIDE 7

Figure 11.15

Ampicillin resistance Promoter ESM CEN Promoter Polylinker (cloning site)

t/pa

SLIDE 8

Figure 11.20

Polylinker M13 genomic DNA lacP lacZʹ″ ʹ″ Phage in clear plaques have cloned DNA Phage in blue plaques do not have cloned DNA

EcoRI KpnI XbaI SalI PstI HindIII SmaI BamHI

SLIDE 9

X-‑gal ¡Cloning ¡Results ¡

SLIDE 10

Three ¡major ¡types ¡of ¡ ¡ restric8on ¡endonucleases ¡

SLIDE 11

Restric8on ¡endonucleases ¡ produce ¡blunt ¡or ¡s8cky ¡ends ¡

Type ¡II ¡enzymes ¡cut ¡at ¡ palindromic ¡sequences ¡

SLIDE 12

Restric8on ¡endonucleases ¡cleave ¡DNA ¡at ¡specific ¡ sites ¡

SLIDE 13

Restric8on ¡maps ¡

Figure ¡5.18ab ¡

Photo ¡courtesy ¡of ¡FOTODYNE ¡Incorporated. ¡

SLIDE 14

Construc8ng ¡a ¡restric8on ¡map ¡

Figure ¡5.19ab ¡

SLIDE 15

Figure 11.5

Foreign DNA Sticky ends Vector Cloned DNA Introduction of recombinant vector into a host Cut with restriction enzyme Add vector cut with same restriction enzyme Add DNA ligase to form recombinant molecules

SLIDE 16

Figure 11.9

SLIDE 17

cDNA ¡synthesis ¡by ¡ ¡ reverse ¡transcriptase ¡

Figure ¡5.45 ¡

SLIDE 18

Figure 11.14

Plunger Helium gas Gas vent Disc Microprojectiles with transfecting nucleic acid Fine screen Rough screen Target tissue

Before gas release After gas release

SLIDE 19

Figure 11.6 Transformant colonies growing on agar surface X-ray film Positive colonies Replica-plate onto membrane filter Partially lyse cells; add specific antibody; add agent to detect bound antibody in radiolabeled form Lyse bacteria and denature DNA; add RNA or DNA probe (radioactive); wash

ut unbound radioactivity

Autoradiograph to detect radioactivity

SLIDE 20

Polymerase ¡chain ¡ reac8on ¡(PCR) ¡ to ¡amplify ¡DNA ¡

Figure ¡5.31 ¡

SLIDE 21

Southern ¡bloHng ¡is ¡used ¡ to ¡detect ¡specific ¡DNA ¡ fragments ¡

Figure ¡5.22 ¡