Biology for the Nanotechnology Classroom. Erinn Mee 10/9/2015

Transcription

1 Biology for the Nanotechnology Classroom Erinn Mee 10/9/2015

2 Size and Scale

3 Visualizing Bacteria Escherichia coli LM 1000x Escherichia coli SEM 20,000x

4 Visualizing Host-Virus Interactions Scott Kachlany, Cornell University Ithaca, New York, USA and The MicrobeLibrary Tortora et al Microbiology: An Introduction 11 th ed. Pearson Dubrovin et al Atomic Force Microscopy Investigation of Phage Infection of Bacteria. Langmuir. 24 (22), pp

5 Visualizing Eukaryotic Cells Bacteria Homo sapiens Buccal cells Nucleus Eukaryotic cell LM 400x LM on the AFM with cantilever tip

6 Biology in Nanotechnology Nanomedicine Drug Delivery Diagnostics Bionanotechnology / Nanobiotechnology Recombinant DNA Expression Protein purification Biofuels

7 SAFETY FIRST!!! CDC (and OSHA) guidelines Personal protective equipment: gloves, lab coat, goggles, and so forth Laboratory DO NOTs Proper disposal

8 Culture and Subculture of Mammalian Cells

9 Tools of Mammalian Cell Culture BioSafety Cabinet (BSC) Consumables such as media, containers, and pipets CO 2 Incubator

10 Effect of Nanoparticles on Mammalian Cells TEM of silver nanoparticles with diameters of 20 nm (Aldrich Prod. No ), 60 nm (Aldrich Prod. No ), and 100 nm (Aldrich Prod. No ) respectively. Scale bars are 50 nm.

11 Effect of Nanoparticles on Mammalian Cells standard gold nanoparticles for lateral flow

12 Cloning in Mammalian Cells as a Model for Drug Delivery: Liposomes Doxil, Lipodox, Caelyx, and Myocet used to treat different forms of cancer Liposome (1999) by Kosi Gramatikoff from Mimic this delivery using a cationic lipid delivery system such as Lipofectamine

13 PCR and Real-Time PCR Volumes for 1 reaction 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.

14 Real-Time PCR Roche Lightcycler 96

15 Quantitating Gene Expression Roche Lightcycler 96

16 Testing for Microbial Load 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), drinking-water-protection-arewe-doing-it-right-0001

17 Endpoint Genotyping 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 For individuals with a functional copy of the taster gene, broccoli has a bitter taste

18 Endpoint Genotyping Isolate DNA phism%20lab%20manual%20-%20carolina%20biological.pdf

19 Endpoint Genotyping

20 Endpoint Genotyping HEX: Taster FAM: Nontaster

21 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 World Journal of Pharmaceutical Research. Vol 3, Issue 3, )

22 Preparation of Plasmid and Transformation Clone a thermostable alphaamylase gene from Geobacillus stearothermophilus 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

23 Selection and Screening of Clones Select for transformed cells using ampicillin Screen for amylase production contains ampicillin and starch ampicillin ampicillin kimedia.org/wiki/fil e:replica-dia-w.svg ampicillin ampicillin unc=displayarticle&aid=277

24 Benchtop Protein Production and Purification Functional Assay ELISA Western Blot

25 Pilot Scale Batch Production and Purification Pilot Scale Production of Amylase (from E. coli) or tissue Plasminogen Activator (from CHO cells) Preparing equipment Introducing organisms Managing the software Maintaining culture Harvesting organisms

26 Biofuels

27 Questions?