Phillips Group Homepage Caltech Website Home About Us Contact Us The Size of Things The Rate of Things DNA Science Advanced Class Projects Bootcamp 2005 Bootcamp February 2006 Bootcamp October 2006 Bootcamp June 2007 Bootcamp September 2007 APh162 2006 APh162 2007

Tethered Particle Motion

Stephanie Johnson, Geoffrey Lovely, David Schwab, Sung Wook Woo

Tethered Particle Motion (TPM) uses the Brownian motion of a polystyrene bead, visible through light microscopy techniques, as a reporter of the motions of single molecules, which are of course not visible under light microscopes. In our case the bead is attached via specific small-molecule interactions to one end of a DNA molecule whose other end is attached via different small-molecule interactions to the surface of a glass coverslip, as shown below.

The extent of the bead's motion is related to the length of the DNA molecule: longer DNA's will allow a greater radius of motion of the attached bead, while shorter DNA's will make this radius of motion smaller. Thus TPM allows us to study processes that alter the length of the DNA "tether" by effecting changes in its conformation. Custom Matlab software allows us to monitor the Brownian motion of the bead as a function of time ("RMS"), giving a readout in real time of the DNA configuration; we can also histogram the RMS motion to clearly observe distinct states:

The September '07 Bootcamp TPM project focused on the formation of loops in the DNA by the Lac Repressor protein, which regulates transcription of the lactose operon in E. coli. If two or more repressor binding sites (operators) are present on a DNA molecule, the repressor can bind two operators simultaneously for a certain length of time, forming a loop in the DNA that effectively shortens the "tether" and therefore reduces the bead's radius of motion. Previous work in the Phillips lab had characterized the effects of operator spacing, loop sequence, and repressor concentration on two-operator systems such as the one shown above. However, in E. coli there are actually three operators present, i.e. the in vivo system has the potential to form three different loops. Biochemical work has shown that having two sites present increases the efficacy of the repressor's action precisely because looping can occur; but it has also shown that three operators is even better than two, for unknown reasons (Oehler et al, 1994). During the September '07 Bootcamp the TPM group examined looping in the three-operator system, with the in vivo loop spacings and loop sequences, and at a variety of concentrations similar to those found in vivo, with the exciting result that concentration seems to play an important role in which of the three loop dominates the system.

The final presentation given on the last day by the students working on this project is shown here:


Copyright Phillips Group 2005-2008