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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
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
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.
Useful Links
Important Model Organisms
Protocols and Handouts
- Week 1 - Microscopy and Optics & Size of Things
- Microscopy and Optics
- Size of Things
- Week 2 - Rate of Things
- DNA Science
- 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
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
- 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.
- 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.
Student Webpages
- The Rate and the Size of Things
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
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