Which plant is transgenic? - PowerPoint PPT Presentation

Loading...

PPT – Which plant is transgenic? PowerPoint presentation | free to download - id: 84507f-OTk3Z



Loading


The Adobe Flash plugin is needed to view this content

Get the plugin now

View by Category
About This Presentation
Title:

Which plant is transgenic?

Description:

Title: PowerPoint Presentation Author: Linda Hanley Last modified by: Dennis Created Date: 4/26/2010 4:38:13 PM Document presentation format: On-screen Show (4:3) – PowerPoint PPT presentation

Number of Views:57
Avg rating:3.0/5.0
Slides: 25
Provided by: Linda743
Learn more at: http://biochem.ncsu.edu
Category:
Tags: plant | transgenic

less

Write a Comment
User Comments (0)
Transcript and Presenter's Notes

Title: Which plant is transgenic?


1
Which plant is transgenic?
  • Determination using an
  • enzyme-linked immunosorbent assay

2
transgenic plants / GMOs
  • A transgenic plant has incorporated gene(s) from
    another species.
  • Transgenics are useful for engineering a
    specific phenotype or studying a genes function.

3
transgenic selection NPTII
  • Selection of plants that incorporated the
    transgene can be performed by simultaneously
    introducing a drug resistance gene into the plant
    (just like we do with E.?coli).
  • nptII (most common) codes for the enzyme
    aminoglycoside 3'-phosphotransferase (NPTII) that
    phosphorylates (inactivates) aminoglycoside
    antibiotics including kanamycin, neomycin
    geneticin (G418) and paromomycin.

4
transgenic selectionkanamycin resistance due to
NPTII
kanamycin
WT
kanamycin
Transgenic NPTII
5
Which plant is transgenic?
6
Which plant is transgenic?
  • Need both WT and transgenic plants for experiment
    (today) so kanamycin selection is out.
  • Perform test for transgene or transgene protein?
  • ELISA is a fast way to test for the presence and
    amount of a protein such as NPTII.

7
ELISAunderstanding the acronym
  • EL Enzyme-linked
  • An enzymes activity is used as a reporter to
    test for the presence/amount of a protein of
    interest.
  • The enzymatic reaction will produce a colored
    species.

8
ELISAunderstanding the acronym
  • EL Enzyme-linked
  • IS Immunosorbent
  • An antibody or antigen (immuno) is adsorbed
    (sorbent) onto the polystyrene wells in which
    we conduct the test.
  • One antibody is already adsorbed added to the
    wells and a second antibody with our enzyme will
    be added in the type of ELISA we will perform
    today.

9
ELISAunderstanding the acronym
  • EL Enzyme-linked
  • IS Immunosorbent
  • A Assay
  • We will could determine the amount of NPTII
    (quantitative assay).
  • We will use NPTII concentration standards to
    construct a standard curve.

10
ELISA uses antibodies
  • What is an antibody?
  • What types of antibodies exist?
  • What kinds of antibodies are used in our lab
    today?

11
What is an antibody?
  • Protein secreted by B-cells that specifically
    bind a foreign substance (antigen)
  • Immunoglobulin domains
  • Complementarity-determining Regions (CDRs)
  • Fab Fragment antigen binding
  • Hinge
  • Fc Fragment crystalline
  • F(ab)2 Protease digestion still useful to bind
    antigen

12
producing polyclonal antibodies
13
producing monoclonal antibodies
1
2
3
14
Antibody-based assays
15
Types of immunodetection systems
2. Indirect immunodetection Secondary
antibody conjugated with enzyme system
1. Direct immunodetection Primary antibody
conjugated with enzyme system
HRP
HRP
HRP
HRP
Ag
Ag
Ag
Ag
3. Sandwich indirect immunodetection
Antigen applied in soluble form
4. Indirect immunodetection with biotin
linkers Biotinylated primary antibodies
HRP
HRP
HRP
HRP
Ag
Ag
Streptavidin
Ag
Ag
16
Simple ELISA protocol(For screening monoclonal
antibodies)
1. Coat antigen onto microplate
2. Allow protein adsorption and block unoccupied
sites with neutral protein
3. Add antibody solution into each well
4. Add enzyme conjugated secondary antibody into
each well and develop colorimetric reaction
with appropriate substrate
5. Read absorbance in spectrophotometer
17
Todays assay
18
Sandwich indirect immunodetection
  • Antigen applied in soluble form

NPTII
NPTII
19
Sandwich ELISA protocol
1. Coat primary antibody onto microplate.
1a. Allow antibody adsorption and block
unoccupied sites with neutral protein
(BSA).
2. Add antigen sample to be detected into each
well. Incubate 30 min at 370 C.
3. Add second primary antibody against antigen
and HRP-conjugated secondary antibody (antibody
mix) into each well. Incubate 30 min at 370 C.
4. Develop colorimetric reaction with
appropriate substrate. Incubate 15 min at room
temperature.
  • Stop reaction with 3M H2SO4. Read
  • absorbance in ELISA spectrophotometer
  • and quantitate relative antigen levels.

20
What you will do today (1)
  • Collect and weigh tissue sample plant A, B and C.
  • Repeat for second and third plants.
  • Add 400 µL PEBX1 buffer to each microcentrifuge
    tube and grind plant tissue using pestle.
  • Add 100 µL of PEBX1 to wells A and B.
  • Add 100 µL of one sample to wells C and D.
  • Add 100 µL of second sample to wells E and F.
  • Add 100 µL of third sample to wells G and H.
  • HANDLE THE WELLS LIKE A CUVETTE (read at bottom)

A B C D E F G H
21
What you will do today (2)
  • Incubate 30 minutes at 37 C.
  • Place wells in a zip-close bag to prevent them
    from drying out in the oven.
  • Wash wells 3 times with PBST
  • Add antibody-enzyme conjugate (MRS-2 Ab)
  • Note the dilution factor for the conjugate
  • Incubate 30 minutes at 37 C.
  • Place wells in a zip-close bag to prevent them
    from drying out in the oven.
  • Wash 4 times with PBST.

22
What you will do today (3)
  • Add substrate and allow blue color to develop.
  • Stop the enzymatic reaction with 3M H2SO4.
  • Presence of NPTII will result in color change
    from blue to yellow.

23
How we will detectread absorbance at 450 nm
24
Data
    Absorbance 450 nm Absorbance 450 nm Absorbance 450 nm Absorbance 450 nm Absorbance 450 nm Absorbance 450 nm Absorbance 450 nm Absorbance 450 nm Absorbance 450 nm Absorbance 450 nm Absorbance 450 nm Absorbance 450 nm
    1 2 3 4 5 6 7 8 9 10 11 NPTII Std
W E L L A                      
W E L L B                      
W E L L C                      
W E L L D                      
W E L L E                      
W E L L F                      
W E L L G                      
W E L L H                      
25
Data analysis (due 4/23/12)
    Total amount of plant tissue (mg) A450 NPTII concentration (ng/mL) NPTII amount (ng protein /mg tissue) Transgenic? A 450 NPTII concentration (ng/mL)
    Your Sample Your Sample Your Sample Your Sample Your sample Standard Standard
W E L L A Blank N/A  0 N/A N/A 2
W E L L B Blank N/A  0 N/A N/A 1
W E L L C Plant __   0.5
W E L L D Plant __  Average Average Yes/No 0.25
W E L L E Plant __   0.125
W E L L F Plant __  Average Average Yes/No 0.0625
W E L L G Plant __   0.03125
W E L L H Plant __  Average Average Yes/No 0.015625
About PowerShow.com