biology for the nanotechnology classroom
TRANSCRIPT
Size and Scalehttp://learn.genetics.utah.edu/content/cells/scale/scale.html
Visualizing Bacteria
Escherichia coli SEM 20,000xEscherichia coli LM 1000x
Visualizing Host-Virus Interactions
© Scott Kachlany, Cornell University Ithaca, New York, USA and The MicrobeLibrary
Tortora et al “Microbiology: An Introduction” 11th ed. PearsonDubrovin et al. 2008. “Atomic Force Microscopy Investigation of Phage Infection of Bacteria.” Langmuir. 24 (22), pp 13068–13074
Visualizing Eukaryotic Cells
LM on the AFM with cantilever tip
Homo sapiensBuccal cells
LM 400x
Eukaryotic cell
Nucleus
Bacteria
Biology in NanotechnologyNanomedicine
• Drug Delivery
• Diagnostics
Bionanotechnology / Nanobiotechnology
• Recombinant DNA Expression
• Protein purification
• Biofuels
SAFETY FIRST!!!• CDC (and OSHA) guidelines
• Personal protective equipment: gloves, lab coat, goggles, and so forth
• Laboratory DO NOTs
• Proper disposal
Culture and Subculture of Mammalian Cells
Tools of Mammalian Cell Culture
BioSafety Cabinet (BSC)
Consumables such as media, containers, and pipets
CO2 Incubator
Effect of Nanoparticles on Mammalian Cells
TEM of silver nanoparticles with diameters of 20 nm (Aldrich Prod. No. 730793), 60 nm (Aldrich Prod. No. 730815), and 100 nm (Aldrich Prod. No. 730777) respectively. Scale bars are 50 nm.
Effect of Nanoparticles on Mammalian Cells
http://www.cytodiagnostics.com/store/pc/Standard-Gold-Nanoparticles-for-Lateral-Flow-c246.htmstandard gold nanoparticles for lateral flow
Cloning in Mammalian Cells as a Model for Drug Delivery: Liposomes
Mimic this delivery using a cationic lipid delivery system such as Lipofectamine
Liposome (1999) by Kosi Gramatikoff from https://en.wikipedia.org/wiki/Nanomedicine
Doxil, Lipodox, Caelyx, and Myocet used to treat different forms of cancer
PCR and Real-Time PCR
Reagent Quantity
Green Master Mix 10 µl
10 µM Primer 1 1 µl
10 µM Primer 2 1µl
DNA 1 µl
Water 7 µl
1. Determine how much master mix, primer 1, primer 2, and water is required for 8 standards, 2 unknowns, 1 negative control, and one “extra” reaction.
2. Correctly assemble these common ingredients, then deliver 19 µl to each PCR tube.
3. Add 1 µl of the correct DNA to each tube. Instead of DNA, add 1 µl water to the negative control.
Volumes for 1 reaction
Real-Time PCR
Roche Lightcycler 96
Quantitating Gene Expression
Roche Lightcycler 96
Yang et al. “Development of a One-Step Immunocapture Real-Time RT-PCR Assay for Detection of Tobacco Mosaic Virus in Soil” Sensors 2012, 12(12), 16685-16694
Testing for Microbial Load
http://www.foodqualitynews.com/Food-Outbreaks/Cross-contamination-risk-from-washing-raw-chicken
http://www.wateronline.com/doc/drinking-water-protection-are-we-doing-it-right-0001
Endpoint Genotyping
• For individuals with a functional copy of the taster gene, broccoli has a bitter taste
• Strong correlation between the ability to taste phenylthiocarbamide (PTC) and single base changes in the gene TAS2R38
• A compound similar to PTC is found in many green vegetables
Endpoint Genotyping
http://abccoolimages.com/cell+dna+extraction
Isolate DNA
http://biomed.brown.edu/arise/resources/docs/PTC%20polymorphism%20lab%20manual%20-%20Carolina%20Biological.pdf
Endpoint Genotyping
Endpoint Genotyping
HEX: TasterFAM: Nontaster
Recombinant Gene Expression in Escherichia coli
• Amylases are enzymes that break down starch and glycogen
• They are found in most organisms
• Used in the food, brewing, textile, detergent, and pharmaceutical industry
• Estimated worldwide market of US $2.7 billion in 2012 with 4% annual increase (Elmarzugi et al. “Amylase Economic and Application Value.” 2014. World Journal of Pharmaceutical Research. Vol 3, Issue 3, 4890-4906)
Preparation of Plasmid and Transformation
Clone a thermostable alpha-amylase gene from Geobacillusstearothermophilus into E. coli
• Purify the gene from G. stearothermophilus and insert into pUC18 or
• Use a premade plasmid (pAMY2014)
Transform E. coli JM109 with the plasmid
http://www.gbiosciences.com/researchproducts/imageview.aspx?imgid=f880051c-ef95-4c6c-b849-358f225f3260
Selection and Screening of Clones
Select for transformed cells using ampicillin
http://inst.bact.wisc.edu/inst/index.php?module=book&type=user&func=displayarticle&aid=277
Screen for amylase production
contains ampicillin and starch
https://commons.wikimedia.org/wiki/File:Replica-dia-w.svg
http://www.geocities.ws/micro2052000/exchangegen.htm
ampicillin
ampicillin
ampicillin ampicillin
Benchtop Protein Production and Purification
http://www.gbiosciences.com/PDF/Protocol/BTNM-9C_Amylase_Ion_Exchange.pdf
Functional Assay
Western Blothttp://www.mdpi.com/2076-2607/3/3/567
http://www.gbiosciences.com/PDF/Protocol/BTNM-6D_Amylase_Direct_ELISA.pdf
ELISA
Pilot Scale Batch Production and Purification
Pilot Scale Production of Amylase (from E. coli) or tissue Plasminogen Activator (from CHO cells)
http://depts.washington.edu/sfrbbl/Facilities.html
• Preparing equipment
• Introducing organisms
• Managing the software
• Maintaining culture
• Harvesting organisms
Biofuels
http://wardsauto.com/news-amp-analysis/global-auto-makers-crossroads-biofuels-world
Questions?