Oxygen: Essential for Eukaryotic life both plants and mammals - - PowerPoint PPT Presentation

Development of Oxygen Sensors in Eukaryotes Oxygen: Essential for Eukaryotic life – both plants and mammals Goal: Develop modular tool for oxygen detection in Euks Oxygen in plants Oxygen in mammals Issue: Finding (or developing) a conserved oxygen sensing system in both plants and animals Divergence – decided to manipulate existing cellular mechanisms to develop unique and different ways of sensing in each type of organism Introduce plant part and functionality Introduce mammalian part and functionality Design selection: researching and evaluating appropriate method for oxygen (hypoxia) sensing Determination of appropriate indicator for oxygen levels – modulate sensitivity? Lab work – transformation and insertion of parts into systems of interest Converge - Experiments - determining functionality of parts over range of conditions Applications /Future Work – develop models for pathological conditions, etc. Ethical Review –genetic modification of eukaryotes in general; plants in specific

The Idea: Oxygen Sensing for Eukaryotes

Function of Oxygen in Eukaryotes

• Final electron acceptor in Cellular Respiration for Aerobic Metabolism
• Determines the progression of respiration
• Essential for harnessing energy in the cell
• Influences all energy-requiring reactions of the cell

Image Source: biologycorner.com

Problems in Oxygen Availability

• Waterlogged plants and

Anaerobic fermentation

• Energy Availability
• Effects on the plant

– Senescence, chlorosis, stunting, death

• Mammalian cells – halts

metabolism

• Molecular signaling cascade
• Effects on the mammal:

– Cell death, fermentation, vasculogenesis/angiogenesis

Image Source: Heddleston, et al. 2009

Objective: Develop Oxygen Sensitive Biological Devices

• Subsequent change

in cellular activity

• Responsive to low
• xygen levels
• Modular device

Problems in Developing Modular Oxygen Sensing Devices

• Incompatibility between

plants and animals

• Evolutionary divergence
• Complex pathways
• Diverse responses to the

same stimulus

Analogous Devices for Oxygen Sensing in Plants and Animals

Plant Device

• Alcohol dehydrogenase (ADH) promoter
• Nuclear localization sequence, Kozak

sequence

• Reporter protein (fluorescent protein,

luminescent protein)

Mammalian Device

• Constitutive promoter (CMV)
• Nuclear localization sequence, Kozak

sequence

• Protein domain – oxygen dependent

degradation

• Reporter protein (luminescent)

Divergence in Paths

Plants Mammals

Image Source: http://pested.ifas.ufl.edu/

200 μm

Model : A. Thaliana Model: Immortalized human GBAM1 cell line (stem cell-like glioma cells)

Plants: Development Criteria

• Why ADH promoter?

– Well-characterized in literature – Up-regulated in hypoxia – Directly involved in respiration pathway – Other options less feasible

Image Source: Meuser, J. 2004.

Plants: Indicating Oxygen Concentration

• Choice of reporter

protein:

– Fluorescent proteins – easily visualized in roots – Luminescent proteins (luciferase) require luciferin substrate – Practical quantification

Image Source: WikiVisual

Plants: Assembly and Insertion of Device

• A. Thaliana cultured on agar plates
• A. Thaliana grown in mist room pots

3A and standard assembly Transforming Process for A. Thaliana : Agrobacterium tumefaciens DNA transferred to agrobacterium via electroporation

• A. thaliana

grown until flowering (30 days) Floral dip

Plants: Determining Oxygen Sensitivity

• Experiments to

include:

– Plant response to low

• xygen, varying time,

concentration – Quantifying GFP production at different oxygen concentrations – Model development

Image Source: biospherix.com

Mammals: Development Criteria

• Why a degradation

domain?

– Limits undiminished signal – Mimics the activity of proteins involved in hypoxia response – Constitutively up- regulates reporter – Terminates response in normoxia – Real time hypoxia sensor

Image Source: dbs.umt.edu

Mammals: Indicating Oxygen Concentration

• Choice of reporter

proteins

– Bright, measurable signal – Luciferase (bioluminescence) - tissue penetration – Easily visualized – Luciferin in cell media

Preliminary transformations using luciferase

Mammals: Assembly and Insertion of Device

• Synthesized active sequence of
• xygen dependent degradation

(ODD) domain

• 3A assembly – modified with

protein fusion

• For insertion into mammalian

cells – transfection by electroporation

Confirmation of Parts for assembly -

Mammals: Determining Oxygen Sensitivity

• Experiments to include:

– Incubation at various

• xygen concentrations

– Expression in differentiated versus proliferative cells – Model to predict reporter activity – Modeling hypoxia in 3D solid tumor cultures

Image Sources: Dirks, Peter B. 2004

Applications

• Crop stabilization –

food production

• Physiological

conditions in plants

• Predictive models

for cancer hypoxia

• Spatial models –

tissue engineering

Image Source: venturebeat.com

Ethical Review

• Weedy or invasive species
• Reductionist philosophy
• Homogenization of plant species
• Uncontrolled transfer of genetic material

• Binary vector systems

– Formerly, transforming agrobacterium meant recovering, manipulating, and reincorporating the tumor-inducing plasmid – In a binary vector system, the plasmid containing the DNA of interest can be manipulated and copied in the more robust E. coli – The vector can then be transferred via electroporation to an agrobacterium with virulence genes on a separate plasmid

• Agrobacterium strain GV3101::pMP9

– Disarmed strain; does not produce tumors – Expresses rifampicin resistance genes – Binary vector pCB302 – Many advantages for plant transformation:Includes antibacterial (kanamycin) and herbicide (glufosinate ammonium) resistance for selection on appropriate media