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Revision as of 13:47, 6 May 2009
20.109 Research Proposal for Derek Ju and Alvin Chen
Our goal is to rewire a bacterium to "miniprep" itself, when given some sort of an external stimuli (such as sensing of dense cell growth). This project will serve the following purposes:
- Make the research process easier, cheaper, and more efficient for scientists working with bacteria.
- Most laboratory strains have been genetically modified for scientific use; however our bacteria would be the first to be modified to do an experiment on itself.
- Self-lysing of cells when dense cell growth is encountered - less chance for environmental contamination if genetically engineered bacteria escape from the lab
- Serve as an example of a way synthetic biology can be used for cellular control
- Be an application of using irreversible switches within genetic circuits
- Be an example of sensors being built into bacteria
Research Problems and Goals
Project Details and Methods
In order to complete our project, we will attempt to incorporate several new genes into E. coli, which will encode for various proteins.
- We will insert a gene encoding for a binding protein that will be attached to the membrane of the bacteria. This protein will most likey contain leucine zippers and as a result the bacteria will lump together in culture.
- The attachment of the binding proteins to eachother will indicate clumping of cells, which will release a signal to toggle a "genetic switch". This switch will be a collection of genes, which when combined creatively, can have an input and output.
- The activation of the genetic switch will lead to expression of a gene encoding for lysozyme, which will lyse the cell.
- The activation of the genetic switch will lead to expression of a gene encoding for RNAse A, which will degrade the RNA in the cell.
- Appropriate DNA sequences for genes, signal sequences, etc
- Standard plasmid for transformation into E. coli, such as pSK1A2
- Some of the DNA for the genetic switches can be retrieved from the Biobricks foundation
- Proteins bind to promoter regulatory sites to repress or activate expression. This results in two stable states: proteins (repressor) repress their own repressor, so they will remain stably expressed. But, switching is controllable switching: dominant repressor (state) can be de-activated with addition of inducer (IPTG, thermal shock, etc). This allows expression of the other repressor, and enables controllable state change of the system
- Leucine zippers are a common structures that have key functions within binding proteins.