Research Proposal

Project Idea
Selection of ethanol and sugar tolerant E. coli from a library of Hsp90 mutants.

A library of Hsp90 mutants will be constructed with ePCR. SDM mutants may be made if there are any particularly compelling regions that we can specifically identify to mutate. The rationale is that more promiscuous Hsp90, or more actively expressed Hsp90 (or non-degraded), will be able to buffer a larger amount of phenotypic variation than standard strains. This underlying abnormally large phenotypic diversity will be exposed in subsequent screens of growth under industrially relevant concentrations of sugar and ethanol. The rate of succesful mutants will be compared to standard random mutagenesis techniques. Particularly succesful mutants will be sequenced and undergo DNA microarray analysis, to determine the gene clusters up or downregulated in the mutants.

References

'''Alper, H., and Stephanopoulos, G. Global transcription machinery engineering: A new approach for improving cellular phenotype. Metab. Eng. 2007; 9:258–267.'''

This paper shows how global modifications to the transcriptome can be made by engineering the cellular transcription machinery. In particular, s70 is modified using this technique of global transcription machinery engineering (gTME) which allows multiple gene modifications to be made simultaneously. This would be very useful when engineering phenotypes which are affected by many genes. This paper will also serve as a reference for a detailed protocol for phenotype selection (ethanol tolerance, for example).

'''Queitsch C., Sangster T., Lindquist S. Hsp90 as a capacitor of phenotypic variation. Nature 2002; 417:618-624.'''

This paper talks about Hsp90's role in buffering genetic variation and how reducing Hsp90 function resulted in the observance of a variety of morphological phenotypes. This paper helps us identify how making modifications to Hsp90 can produce different phenotypes, given its role in chaperoning regulatory proteins, buffering genetic variation and influencing responses to environmental changes and stress.

'''Whitesell L, Lindquist S. HSP90 and the chaperoning of cancer. Nature Reviews Cancer 2005; 5:761-772'''

This paper provided us with more background on HSP90's chaperoning functions and its role in cancer. It talks about the function and structure of HSP90 in detail and will help us identify particular regions to mutate if we chose to do so.

'''Sangster T.A., Salathia N., Undurraga S., Milo R., Schellenberg K., Lindquist S., Queitsch C. HSP90 affects the expression of genetic variation and developmental stability in quantitative traits. Proc. Natl. Acad. Sci. USA 2008 105:2963-2968.'''

This paper is another source discussing the role of HSP90 in concealing genetic variation. The hypothesis discussed is that changing HSP90 function may lead to the evolution of new phenotypes in response to environmental stress. They checked to see changes in hypocotyl elongation in the dark in Arabidopsis thaliana. We want to take this idea further to check to see changes in ethanol and sugar tolerance after HSP90 modification. The general idea is that the revelation of poymorphisms previously buffered by HSP90 may lead to some interesting evolutionary behavior in suboptimal environments. Another thing to note from this paper is that they found that changes in the traits they were looking at outweighed decrease in developmental stability (affected  by the natural variation buffered by HSP90 as well).