Lecture 11 Chapter 7 Vector Construction II

1
Lecture 11 Chapter 7Vector Construction II

• Gateway cloning
• David Mann

2
What is cloning?
3
http//upload.wikimedia.org/wikipedia/commons/thum
b/6/66/Scissors.svg/540px-Scissors.svg.png
4
So, youve 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
Klenow Fragment
Ligase
8
Site-specific RecombinationGateway Cloning
9
Bacteriophage ?
10
Bacteriophage ?
11
Figure 7.11
12
(No Transcript)
13
Gateway cloning
Figure 7.12
14
Figure 7.13
15
Transform into DH5a E. coli cells
16
Transform into DH5a E. coli cells
http//media.invitrogen.com.edgesuite.net/presenta
tions/gateway/index.html?icidfr-gwcloning-7
17
(No Transcript)
18
Site-specific RecombinationCreator Cloning
19
Cre/loxP Recombination System
The Jackson Laboratory (http//www.jax.org/index.h
tml)
20
Creator cloning
Figure 7.14
21
Figure 7.15
22
Figure 7.16
23
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

24
Conventional cloning
Gateway cloning
Site-specific DNA recombination
Creator cloning Univector cloning
Figure 7.17
25
RNA interference pathway
26
Vectors for RNAi
Figure 7.18
27
Figure 7.21
28
Figure 7.22
29
Multisite Gateway allows several DNA fragments to
be cloned into a single construct
Figure 7.19
30
Katzen 2007. Expert Opin. Drug Discov. 2(4)
571-589
31
Gebert et al., The Plant Cell, Vol. 21
40184030, December 2009
Multiple promoters from the MRS2/MGT gene family
fused to the GUS gene and expressed in
Arabidopsis thaliana
32
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

33
Figure 7.20
34
(No Transcript)
35
Figure 7.23
36
Example of a plant expression vector set

• pANIC
• Made for switchgrass transformation
• BioEnergy Science Center (BESC)
• http//plantsciences.utk.edu/stewart.htm

37
The pANIC vector set

• Functional in switchgrass and rice
• Overexpression (OE) and suppression (RNAi) of
  genes
• ZmUbi1
• CaMV 35S
• Protein tag for OE AcV5

RNAi of lignin biosynthetic genes in switchgrass
Shen Dixon, Noble Foundation
37
Mann et al. Plant Biotechnology Journal 2012
38
pANIC - Reporter cassette
GUS staining photos courtesy of Zach King

• PvUbi1 promoter
• Histochemical
• GUSPlus
• Fluorescent
• pporRFP novel RFP
• Comparable to DsRed
• Ex/Em 578 nm/595 nm

Brightfield 5ms
RFP 2s
GFP 10s
DsRed
pporRFP
39
pANIC Vectors