Biology for the Nanotechnology Classroom

Biology for the Nanotechnology Classroom

Erinn Meeerinnmee@oakton.edu

10/9/2015

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?