Imaging of Rotavirus

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Imaging of Rotavirus with Atomic Force
Microscopy Construction of fluorescently
tagged Acs protein Summer Lab Rotation
presentation By Sangeetha Rajagopalan GST
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Atomic Force Microscope (AFM)

• Scanning Probe Microscopy (SPM) family of
  microscopes where a sharp probe (tip) is scanned
  across a sample (tip-scanning system) or a sample
  is scanned across a tip (sample-scanning system)
  at nano-scale distances to detect interactions
  between the tip and the sample
• SPM
• AFM
  STM
• (Atomic Force Microscopy)
  (Scanning Tunneling Microscopy)

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PicoPlus Scanning Probe Microscope
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AFM

• AFM - device used to image materials at the
  atomic level
• AFMs are used to solve processing and materials
  problems in electronics, telecom, biology and
  other high-tech industries
• Invented by IBM in 1986
• AFM images information about surface features
• Can examine any rigid surface in air or with
  the specimen immersed in a liquid
• Field of view vary from atomic and molecular
  scale up to sizes larger than 125µm
• No dehydration of sample necessary

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Components of the AFM system

• Computer runs Molecular Imaging System through
  PicoScan software
• PicoScan controller contains the analog
  electronics, ADC DAC boards to generate
  process various signals such as servo and
  scanning voltage
• AC mode controller generates either AAC or MAC
  mode drive signal depending on the mode of
  operation
• Head Electronics processes and communicates the
  signals between the microscope and the controller
  depending on the configuration of the system

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Components of AFM

• Microscope consists of piezoelectric scanner (
  for imaging), photo detector and sample stage,
  sharp tips, flexible cantilevers
• the tip and cantilever tip mounted on end of a
  small cantilever
• tip in close contact with sample, gives rise to
  images though its force interactions with the
  surface
• tip-cantilever assembly, fabricated from silicon
  or silicon nitride
• Essential parameters in a tip radius of
  curvature the aspect ratio
• Four types of cantilevers type I, II, III IV
  classified by their force (or spring) constant
  resonance frequency
• deflection sensor AFMs use optical lever or
  beam-bounce method to
  measure vertical deflection of the cantilever
• scanner accurate positioning stage, used to
  move the tip over sample, made from a
  piezoelectric tube

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PicoPlus AFM
Top Down Scanner
Stage
Heating/Cooling Stage
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Operation of AFM

• AFM images recorded using PicoSPM scanning probe
  microscope
• Operates in MacMode non-contact mode of
  operation
• MacMode operates by placing a solenoid beneath
  the sample establishing an AC field to excite a
  magnetically treated cantilever into oscillation
  at its resonance frequency
• Proximity of cantilever tip to the surface
  reduction of oscillation amplitude
• As the tip is moved over the material, it
  periodically senses the surface and bends as it
  is repelled or attracted to the structure
• Laser beam reflected from cantilever onto
  photodiode, identifies deflections, determines
• height

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Preparation of mica surface and sample for AFM

• Circular mica disks punched out of a flat mica
  sheet
• Disks cleaved to obtain thin individuals disks
• Disks coated with solution containing 0.5
  gelatin 0.01 chromium ammonium sulphate the
  solution heated to 600C
• The disks were dipped in the solution and dried
  overnight
• 1µl of rotavirus sample was taken and diluted
  with 49µl of sterile distilled water
• The diluted sample was spotted on the mica disc
  and allowed to stand for 10-20 minutes
• Rinsed the disc with deionized water and dried
  for imaging in air or covered with distilled
  water for liquid imaging
• The disc was mounted on to the sample holder and
  imaged using type II cantilever with spring
  constant 2.8N/m using MacMode at a scan rate of
  1.48Hz(1.48 lines/s)

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Purpose of this study

• To immobilize rotavirus on a surface analyze
  its image by AFM
• Study the various outer and inner capsid proteins
  with suitable antibodies coated on to the tip of
  cantilever

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Rotavirus

• Rotavirus primary cause of severe dehydrating
  diarrhea in infants and young children
• Member of the Reoviridae family
• Double-shelled, spoke like appearance electron
  micrographs of negatively stained preparations
• Composed of two capsid layers inside which
  resides a core
• Core 11 double-stranded RNA genome segments
• Structural proteins in core particles VP1, VP2
  VP3
• VP6 major inner capsid protein subgroup
  determinant
• VP7 major outer capsid protein determines
  viral serotype
• VP4 - outer capsid protein manifests
  hemaggulatinin activity, neutralization antigen

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Rotavirus immobilized on mica surface and imaged
with PicoSPM in air
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Rotavirus immobilized on mica surface and imaged
with PicoSPM in air
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Rotavirus immobilized on mica surface and imaged
with PicoSPM in air
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Rotavirus immobilized on mica surface and imaged
with PicoSPM in liquid
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Future work

• The ultimate goal of the project is to immobilize
  the virus on a surface measure the strength of
  binding of different sets of antibodies to the
  different viral capsid proteins
• Work done sample preparation for imaging
• attaching of the viral
  sample to mica surface
• (immobilization of
  virus on mica surface)
• imaging of the
  rotavirus in both air and liquid

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Construction of fluorescently tagged Acs protein
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Study the role of Acs gene in carbohydrate
metabolism in E.coli

• Acetyl-CoenzymeA (acetyl-CoA) - an important
  metabolite in a variety of important
  physiological process that link anabolism and
  catabolism
• Acetyl-CoA essential building block for the
  synthesis of fatty acids, amino acids and biomass
• Conversion of acetate to acetyl-CoA catalyzed by
  acetyl-CoA synthetase (Acs)
• In prokaryotes, there are three distinct pathways
  involved in this conversion
• first pathway comprises two enzymes, the acetate
  kinase and the
  phosphotransacetylase
• second pathway catalyzed by acetyl-CoA synthetase
  (ADP-forming)
• third pathway catalyzed by an AMP-forming
  acetyl-CoA synthetase
• The generated acetyl-CoA can enter TCA cycle and
  generate ATP or be utilized to synthesis of
  biomass.

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Acetyl-CoenzymeA pathway
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Basic steps
Construction of Donor plasmid with Acs gene
GFP
Cotransformation of donor and
mutagenic plasmids into E. coli K12
Genomic integration (Gene gorging)
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Construction of Donor Plasmid

• Amplification of Acs gene with SceI NdeI
  restriction site
• Ligation into PCR blunt vector
• Amplification of downstream region of Acs gene
  using primers with NdeI BamHIxbaI restriction
  sites Acs flank
• Ligation of Acs flank with Acs-PCRblunt
• Amplification of Green fluorescent protein (GFP)
  using primers with restriction sites for NdeI
• Ligation of GFP with Acs-Acs flank-PCR blunt
• Transformation into E.coli Top10 cells
• Check for orientation of insert

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Construction of Donor Plasmid
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PCR analysis of colony of acs PCR blunt
ligation with oilgos M13 forward and 77(Acs
reverse)
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Gene Gorging

• Acs-GFP-Acs flank-PCR blunt donor plasmid
  introduced into bacterial cell (E.coli)
• A mutagenic vector carrying I-SceI endonuclease
  gene and lambda Red gene introduced into the same
  bacterial cell
• The genes under inducible control of the
  arabinose
• Inoculated into arabinose containing medium
  induce I-SceI and Red
• Cleavage of Acs-GFP-Acs flank from PCR blunt by
  SceI enzyme
• In vivo recombination of Acs-GFP-Acs flank with
  bacterial chromosome
• Study of regulation of Acs gene in E.coli under
  metabolic conditions through GFP

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Gene gorging strategy
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Work Done

• amplification of Acs gene
• ligation of Acs gene with PCR blunt
• amplification of Acs flank
• ligation of Acs PCR blunt with Acs flank

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Future work

• This project aims to study the regulation of Acs
  gene under various stress conditions at single
  cell level, by imaging the fluorescently tagged
  Acs protein in E.coli cell
• The described cloning method helps avoid issue of
  high copy number of plasmid and allows to study
  the physiological role of the gene with its
  native promoter
• 
  
  

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Acknowledgement

• Dr. Mitchel J. Doktycz
• Dr. D. P. Allison
• Jennifer L. Morrell-Falvey
• Amudhan Venkateswaran
• Elizabeth T. Owens
• Claretta J. Sullivan
• Nicole Edwards