Programming yeast apoptosis

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a 20.109 Research Project by Jingxun Chen and Elizabeth Choe (Blue group, WF)

Contents

The "Big Picture" Starting Point

  • Our research question: How can the principles of synthetic biology be applied to create effective therapeutics and/or drug delivery systems for cancer treatment?
  • Our starting point is a review article by Shankar and Pillai. (Mol Biosyst. 2011 Mar 24. [Epub ahead of print]. Translating cancer research by synthetic biology. Shankar S, Pillai MR.)
    • The field of synthetic biology aims to manipulate biological parts into higher-ordered, specified systems. In this review article, the authors explain how this methodology is being used in cancer research. Some of the applications they describe are: using directed evolution to develop enzymes that can be used in detection systems, using modules to create drug delivery systems, and using nucleic acids as drug therapies.
    • In particular, we are looking at programmable E. coli or other bacteria that invade tumors
      • If we can manipulate E. coli to safely deliver drugs, what happens after the delivery? How can we safely clear them from the body?
      • Solution: Induce cell death in E. coli after a specific number of cell cycles

Tools: The Synthetic Genetic Counter

Project Problems & Solutions

To-Do List/Technicalities

  • Find guidelines to choose G1 cdk, Promoter X, Molecule A
    • Select a G1 cdk as induction signal for the RTC counter
    • Select a molecule involved in yeast's apoptotic pathway (Molecule A) as the output of the counter
    • Identify a strong promoter (Promoter X) that is acted upon by enzymes downstream of our G1 cdk
  • Implement the constructs in yeast and test them piece-by-piece and as a whole, using transfer functions
    • Swap the sensing promoter in Friedland's RTC counter with Promoter X
    • Replace the GFP reporter with the gene that synthesize Molecule A
    • Test whether the yeast cells undergo apoptosis after three cycles of replication
      • We need to find a way to quantitatively measure the apoptosis (maybe LIVE/DEAD staining like we did in Mod 3?)
      • Keep track of what "cycle" the cells are in - maybe with biotin?
  • Make sure that throughout the process, adding the plasmids doesn't severely lower cell viability

References

1. Apoptosis in yeast 2.

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