User:Mgethers/Research Proposal

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Research Proposal for Jerzy Szablowski and Matt Gethers

Project Overview

We would like to design a method of patterning inorganic materials on the phage coat by regulating populations of p8 that are available for incorporation into the coat.

Background Information

M13 is a filamentous bacteriophage of E.coli approximately 900 nm long and 6.5 nm thick. A complete M13 viral particle consists of a circular positive strand of ssDNA molecule and multiple copies of five types of proteins (Russel, 1991). The protein of our interest is a major coat protein, p8. It exists in approximately 2700 copies on the M13 particle making it a good target for display of useful peptides. Prior to incorporation into viral particle protein p8 has to be localized into the membrane and cut in the middle. Its C terminus localizes in the cytoplasm and the N terminus in the intermembrane space. After inserting into the membrane, p8 is attached to the DNA of M13 that spans the outer and inner membranes of E.Coli. During the attachment of the next subunits of p8, DNA is pushed outside of the cell and thus the following p8 proteins create 'bands' or 'rings' of protein p8 around the DNA. At the very end of the DNA strand, a different protein attaches thus 'capping' M13 and allowing for its dissociation from bacteria.

Problem Definition and goals

Various modifications has been made to M13, so it can be used as a means of presenting a compound with one of the various proteins with different binding affinities (reviewed in Sidhu, 2000). However attempts to incorporate two different p8 mutants in comparable abundances have failed. (Nam et al, 2006). Efficient incorporation of proteins with various binding specificities would allow for constructing an M13 phage that has multiple functionalities, which are applicable in various fields of science such as biomedical imaging. Such particles with multiple versions of p8 would be even more useful if it were possible to arbitrarily pattern p8 proteins on the bacteriophage coat. Then it would be possible to make devices which are smaller than an M13 particle.


Project Details and Methods

During incorporating into M13 coat, one of the mutant p8 proteins is usually strongly favored. To remedy this situation, we decided to control the populations of p8 just prior to insertion. A light-based activation mechanism will allow for simple and versatile control of various populations of p8 available for incorporation into M13.

We will construct two clones of protein p8. Each clone will have a modified sequence at the N terminus that allows the attachment of a photoactivable agent at the terminus. If bulky, such an agent enough could hinder incorporation of p8 into the coat. When the light is shined onto the agent, it would dissociate from the N terminus of p8 leaving it free to attach to DNA and incorporate into the coat. The use of two photoactivable agents that respond to different wavelengths of light would allow the selective incorporation of the p8 mutant proteins.


Predicted Outcomes

This method allows for a 'tricking' m13 coat assembly system by sligtly modifying the existing coat assembly system. This should allow for the incorporation of two populations into the same coat. However, the outcome might be different. The failure may come from the fact that increasing expression levels to incorporate two p8 proteins lowers the phage titer. Bacteriophages with two p8 populations could be inviable. Further research with directed evolution of interfaces of p8 should be done in the case this approach is unsucessful.

Needed Resources

- Artificial aminoacids from King Lab - Various photocrosslinkers and photoactivable agents to test whether they hinder binding to C terminus of p8. They should also be membrane permeable. - A phagemid with one population of p8 that gives a viable m13 and another distinguishable phagemid with p8' giving viable M13 phages.

References

Nam, KT, Kim, D-W, Yoo, PJ, Chiang, C-Y, Meethon, N, Hammond, PT, Chiang, Y-M & Belcher, AM. (2006) Science 312, 885–888.

Sidhu, S. S.; Weiss, G. A. and Wells, J. A. (2000) J. Mol. Biol., 290: 487-495.

Russel, M. Filamentous phage assembly. Mol. Microbiol. (1991), 5, 16071613

http://www.blackwell-synergy.com/doi/pdf/10.1111/j.1399-3011.2004.00203.x?cookieSet=1

http://www.nature.com/nsmb/journal/v5/n10/pdf/nsb1098_891.pdf

http://www3.interscience.wiley.com/cgi-bin/fulltext/108567775/PDFSTART

http://www3.interscience.wiley.com/cgi-bin/fulltext/112510974/PDFSTART

http://intl.pnas.org/cgi/content/full/99/17/11020

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