Programable yeast apoptosis

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

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

• Source: A. E. Friedland, T. K. Lu, X. Wang and D. Shi, et al., Synthetic gene networks that count, Science, 2009, 324, 1199–1202
  • Summary: Synthetic genetic counters in E. coli that can count up to three induction events have been made by Friedland et al. in 2009.
  • This counter is called the riboregulated transcriptional cascade (RTC) counter
  • One potential application of genetic counters is to couple the induction events to cell cycle and induce cell death after user-defined number of cell cycles. Thus, you could theoreticaly "tell" a therapeutic agent to "die" after a specified time.

Project Problems & Solutions

• E. coli does not undergo apoptosis (though there is current research in inducing an apoptosis-like death in bacteria)
  • Solution: Conduct a proof-of-concept project in which the synthetic counter is coupled with apoptosis in yeast, then worry about implementing it in E. coli (probably a totally different project)

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. Note