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APh/BE 162 - Physical Biology Laboratory

Course Instructor: Rob Phillips

[ Course Information | Readings | Protocols and Handouts | Useful Links | Important Model Organisms | Contact Information | Student Webpages | ]

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Image processing results in a wealth of quantitative information; here a lipid bilayer vesicle is deformed using a micropipette to garner information about its mechanical properties. Fourier analysis (blue and red lines) and edge finding (green lines) reveal geometric features of the deformed vesicles for further analysis. (c) Tristan Ursell/Rob Phillips

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Course Information

• Class outline (pdf)
• General Meeting: Wednesday -- 1pm
• Section I: Thursday -- 6 pm to 10 pm, 040 Keck
• Section II: Friday -- 1 pm to 5 pm, 040 Keck

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Readings

• Measuring protein concentration
  
  • Edelhoch H; Spectroscopic Determination of Tryptophan and Tyrosine in Proteins, Biochemistry, Jul 1967; 6(7):1948-54.
    
  • Gill SC and von Hippel PH; Calculation of Protein Extinction Coefficients from Amino Acid Sequence Data, Anal Biochem, 1989; 182:319-326.
    

• DNA Science
  
  • Lutz R and Bujard H; Independent and tight regulation of transcriptional units in Escherichia coli via the LacR/O, the TetR/O and AraC/I1-I2 regulatory elements, Nucleic Acids Research, 1997; 25(6):1203-10.
    
  • Peters JE, Thate TE and Craig, NL; Definition of the Escherichia coli MC4100 Genome by Use of a DNA Array, Journal of Bacteriology, Mar 2003; 185(6):2017–2021.
    

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Useful Links

• Amino Acid Data
• The Protein Databank (PDB)
• Review of 2D Diffusion
• Beer's Law - Tutorial
• Spectra Database - (amino acids, fluorophores, etc)

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Important Model Organisms

• Model Organisms for Biomedical Research @ NIH
• Dictyostelium
• C. elegans
• Drosophila melanogaster

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Protocols and Handouts

• Week 1 - Microscopy and Optics & Size of Things
  • Microscopy and Optics
    • Microscopy and Spatial Calibration (pdf)
    • A brief explanation about fluorescence microscopy (pdf)
    • Some basics on digital imaging and CCD cameras (pdf)
      
      Here are a some papers on the resolution limit
      
    • This is Gustafsson's paper on "Extended resolution fluorescence microscopy" (pdf)
    • Scherer et al. paper on single molecule fluorescence microscopy (pdf)
    • A paper on beating the resolution limit using stimulated-emission-depletion fluorescence microscopy (pdf)
      
      Below we follow with some optics tutorials written by both Dave Wu and taken from Newport
      
    • This is the powerpoint presentation on optics given in class in pdf form
    • This is the detailed tutorial on optics based on the powerpoint presentation (pdf)
    • This is a basic tutorial on how to use lenses in order to focus, collimate, expand, etc. a beam (pdf)
    • A basic tutorial on Gaussian beam optics (pdf)
    • This is a list with brief descriptions about the most commonly used materials in optics (pdf)
    • A list of some commonly used formulas in optics (pdf)
    • A useful list with explanations of commonly used optical terms (pdf)
    • This is an older but condensed version of all the previous Newport tutorials (pdf)
    
    
  • Size of Things
    • Lab Manual "Size of Things" (pdf)
    • Data sheet for the slide of bovine pulmonary cells (pdf)
    • Some useful protocols related to the preparation of samples (media, plates, etc) (pdf)
• Week 2 - Rate of Things
  • Lab Manual "Rate of Things" (pdf)
  • Some useful protocols related to the preparation of samples (media, plates, etc) (pdf)
  • This is the brief powerpoint presentation on particle tracking using Matlab given in class (in pdf form)
  • http://snowdome.caltech.edu/aph162/APh162-2007/Matlab/ Go to this link to access the files that are used at the particle tracking tutorial. You will need to use the class username and password
• DNA Science
  • Week 1: Restriction Digests, PCR and Gel Electrophoresis
    
    • Restriction Endonucleases Assay
    • Invitrogen Accuprime PFX Supermix Protocol
      
    • Useful protocols for working with DNA (pdf)
    • Brief description of all the methods (PCR, transformation, etc.) that we will be using for this part of the course (pdf)
  • Week 2: Gel Extraction, Ligation and Transformation
    
    • Qiagen QIAquick Spin Handbook
      
    • Roche Rapid DNA Ligation Kit Protocol
      
    • Useful protocols for working with DNA (pdf)
    • Brief description of all the methods (PCR, transformation, etc.) that we will be using for this part of the course (pdf)
• Projects
  • DNA Force Extension Using Optical Tweezers (with Dave Wu)
    Over the course of a month, we will build an optical tweezer system in order to measure force-extension of lambda-DNA. We will tether one end of DNA to a surface, and the other end to a micron-sized bead. Then by moving the surface at constant velocity, we will be able to measure the force applied to the bead by the DNA by measuring the bead's position in the optical trap. This will involve some optics for the tweezer itself, and some electronics, as we build the detection systems necessary for determining the position of the trapped bead, and data acquisition and computer interfacing (automation) between instruments. We will also figure out the right chemistries to use in attaching DNA to surfaces, and DNA to beads. Not to mention understanding force-extension properties of DNA. Our group will also attempt to build an autofocus device based on total internal reflection geometry.
    
    
  • Diffusion in Biofilms (with Frosso Seitaridou)
    Bacteria organize themselves in multicellular communities, called biofilms. The bacteria Pseudomonas are producing secondary metabolites, called phenazines, which help the bacteria compete with other organisms in the ecosystem for resources (eg. against fungi, in the case of the roots of crops in the soil ecosystem). Inside biofilms diffusion is the predominant method of transport. However, not much is known about the diffusive properties of phenazines across biofilms. The goal of this project in to grow a Pseudomonas biofilm inside a microfluidics chip and observe the diffusion of phenazines across the biofilm.
    Below, there is some reading material with regards to this project. The papers are mainly divided into two categories: the first one concerns the design, fabrication and applications of microfluidic devices while the second is some background reading on biofilms/phenazines and their properties.
    
    
    • Papers on Microfluidics
      • This paper gives a good overview of the microfluidics fabrication process and some experiments done in order to quantify the use of microfluidics (pdf)
      • This is another paper with a good description of the fabrication process and a good application (pdf)
      • This last one is a long review article which presents all the physics needed to describe microfluidics (pdf)
      • Protocol for the fabrication of microfluidics: How to make molds (pdf)
      • Protocol for the fabrication of microfluidics: How to make chips (pdf)
        
      • Powerpoint presentation on microfluidics (ppt)
        
      In general, Prof. Quake's website at Stanford, http://thebigone.stanford.edu/papers.htm, is a great source of information about the fabrication and applications of microfluidic devices.
      
      
    • Papers on Biofilms and Phenazines
      • A good paper that explains in simple terms what biofilms are and how they manage to thrive (pdf)
      • A paper on the spatial organization of biofilms (pdf)
      • A review paper on the various roles of phenazines for biofilms (pdf)
      • This paper discusses what is known about the process of diffusion in biofilms (pdf)
      • Another paper on phenazines and their interaction with water and DNA bases (pdf)
    
    
  • Gene Expression (with Hernan Garcia)
    The regulation of the expression of genes is key in determining the fate of cells. For example, what makes one of your kidney cells different from the cells in your eyes is that each one is expressing a different set of genes. E. coli is a classic model system for studying gene regulation. In particular, it is common to measure the level of expression of fluorescent proteins off of bacteria (Rosenfeld2005) in single cells using the microscope (as you will all do in class). In this project we will push this technique towards systematic and high throughput quantification of the level of gene expression. The techniques to be used range from flow cytometry to microfluidics combined with microscopy.
    
    Rosenfeld2005 (pdf)
    
    
  • Permeation of Lipid Bilayers using Micropipette Aspiration (with Tristan Ursell)
    Permeation of water and other small molecules through lipid membranes is an important cellular survival process, necessary for regulating chemical concentrations inside the cell, as well as managing osmotic stresses in different regions of the cell. We will be exploring facets of membrane permeation using micropipette aspiration of giant unilamellar lipid vesicles. This entails creating these large lipid structures and their subsequent mechanical/permeability testing in different osmotic environments, as well as with different co-solutes such as salts and sugars. This will be in part a reproduction of the referenced work, however the majority will be new research. Students will be using time-lapsed fluorescence video microscopy, quantitative image analysis, general wet-lab skills, and some theory concerning membrane mechanics in osmotic environments.
    
    
    • Olbrich K, Rawicz W, Needham D, et al. (2000) "Water permeability and mechanical strength of polyunsaturated lipid bilayers", Biophys. J., pp. 321-327, (pdf)
    • Electroformation of Bilayer Vesicles
    
    
    
  • Phagocytosis using macrophages (with Heun Jin Lee)
    We are interested in examining phagocytosis of different sized particles. Phagocytosis refers to the process whereby cells of the immune system known as macrophage ingest antibody coated objects such as E. coli, other dead cells and engineered particles. It has been observed that there is a qualitative difference of the rate of phagocytosis depending upon the size and curvature of the object. The goal of this experiment will be to quantitatively explore this phenomenon and examine the mechanisms for this process.
    
    

• Beta-Galactosidase Kinetic Assay
• Image Analysis with Matlab Tutorial

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Student Webpages

• The Rate and the Size of Things

• DNA Science

• Final Projects

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Contact Information

Rob Phillips
159 Broad
x3374
phillips AT pboc.caltech.edu

Frosso Seitaridou
71 Broad
x5761
frosso AT caltech.edu

Dave Wu
157 Broad
x3106
davewu AT caltech.edu