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Lecture 11 Chapter 7 Vector Construction II

Lecture 11 Chapter 7 Vector Construction II. Gateway cloning David Mann. What is cloning?. http://upload.wikimedia.org/wikipedia/commons/thumb/6/66/Scissors.svg/540px-Scissors.svg.png. Digest vector DNA with restriction enzyme. Gene for blue flowers.

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Lecture 11 Chapter 7 Vector Construction II

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  1. Lecture 11 Chapter 7Vector Construction II Gateway cloning David Mann

  2. What is cloning?

  3. http://upload.wikimedia.org/wikipedia/commons/thumb/6/66/Scissors.svg/540px-Scissors.svg.pnghttp://upload.wikimedia.org/wikipedia/commons/thumb/6/66/Scissors.svg/540px-Scissors.svg.png

  4. Digest vector DNA with restriction enzyme Gene for blue flowers What are essential components of vector DNAs? Ligate gene into vector Plasmid vector Extract plasmid DNA Transform plant So, you’ve cut out a gene… Now what? How did you amplify this gene?

  5. Solving problems of insert orientation

  6. Problems with conventional cloning • Inconvenient restriction sites • Vector construction is laborious • Time-consuming reactions

  7. Blunt-end Ligation PstI PstI EcoRI EcoRI ACGTC C C G G G G G G CTTAA C C Klenow Fragment Ligase

  8. Site-specific Recombination:Gateway™ Cloning

  9. Bacteriophage λ

  10. Bacteriophage λ

  11. Figure 7.11

  12. Gateway™ cloning Figure 7.12

  13. Figure 7.13

  14. Transform into DH5αE. coli cells

  15. Transform into DH5αE. coli cells http://media.invitrogen.com.edgesuite.net/presentations/gateway/index.html?icid=fr-gwcloning-7

  16. Site-specific Recombination:Creator™ Cloning

  17. Cre/loxP Recombination System: The Jackson Laboratory (http://www.jax.org/index.html)

  18. Creator™ cloning Figure 7.14

  19. Figure 7.15

  20. Figure 7.16

  21. Why do we need so many types of vectors? What are some different applications in plants? • Functional analysis of open reading frame (ORFs) • Overexpression and knockdown (RNAi) of specific genes. • Multigenic traits for crop improvement • Analysis of the expression level/specificity/ inducibility of promoters

  22. Conventional cloning Gateway cloning Site-specific DNA recombination Creator cloning Univector cloning Figure 7.17

  23. RNA interference pathway

  24. Vectors for RNAi Figure 7.18

  25. Figure 7.21

  26. Figure 7.22

  27. Multisite Gateway allows several DNA fragments to be cloned into a single construct Figure 7.19

  28. Katzen 2007. Expert Opin. Drug Discov. 2(4): 571-589

  29. Gebert et al., The Plant Cell, Vol. 21: 4018–4030, December 2009 Multiple promoters from the MRS2/MGT gene family fused to the GUS gene and expressed in Arabidopsis thaliana

  30. Vectors derived from plant sequences • Public acceptance of GMOs linked to concerns of the origin of DNA employed • Ironically, wild-type plant cells already contain bacterially-derived genomes • T-DNA could be replaced with P-DNA • Replace viral promoters with plant promoters

  31. Figure 7.20

  32. Pollen genome loxP-FRT loxP-FRT LB RB NOS GUS:NPTII 35S p FLP Recombinase NOS LAT52 Pollen-specific promoter LAT52 activates recombinase in tobacco pollen loxP FRT excision NOS GUS:NPTII 35S p FLP Recombinase NOS LAT52 loxP-FRT LB RB Pollen genome A B Luo et al. 2007. Plant Biotechnology Journal 5(2): 263 - 274. (Courtesy of Moon’s poster)

  33. Figure 7.23

  34. Example of a plant expression vector set • pANIC • Made for switchgrass transformation: • BioEnergy Science Center (BESC) • http://plantsciences.utk.edu/stewart.htm

  35. The pANIC vector set RNAi of lignin biosynthetic genes in switchgrass Shen & Dixon, Noble Foundation • Functional in switchgrass and rice • Overexpression (OE) and suppression (RNAi) of genes • ZmUbi1 • CaMV 35S • Protein tag for OE – AcV5 37 Mann et al. Plant Biotechnology Journal 2012

  36. pANIC - Reporter cassette GUS staining photos courtesy of Zach King Brightfield – 5ms RFP – 2s GFP – 10s DsRed pporRFP • PvUbi1 promoter • Histochemical • GUSPlus • Fluorescent • pporRFP – novel RFP • Comparable to DsRed • Ex/Em = 578 nm/595 nm

  37. pANIC Vectors:

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