Inducing apoptosis by synthetic counter

20.109 Research Project Jingxun Chen and Elizabeth Choe Blue group, WF

The "Big Picture" Question

• 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 this 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.

Detailed Questions

• What are some specific aspects of this field that we could explore?
  • Drug-sensing hydrogels (to fit in with Module 3)
  • Elastin-like polypeptides
  • RNA aptamers that bind to the tumor or deliver therapeutic siRNA (to fit in with Module 1)
  • Programmable E. coli or other bacteria that invade tumors

• What are some of the problems with the current research? (i.e., what needs to be fixed?)
  • Explore this topic more.

Zooming in on the specific problem

• 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 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.
  • Our project: implement Friedland's RTC counter in yeast to induce apoptosis after three replicative cycles

To-Do List

• 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
• 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

Some technical thoughts

• We will also need to:
  • Find guidelines to choose G1 cdk, Promoter X, and Molecule A
  • Test the constructs piece by piece and as a whole (characterized by transfer functions)
  • Assemble the constructs in yeast
  • Ensure that adding the plasmids don't severely lower cell viability
  • Find a way to measure apoptosis in yeast (should have method publications)
  • Label the yeast cells with biotin that indicate the number of division

Elizabeth Choe MIT Department of Biological Engineering || Class of 2013 echoe@mit.edu 573.881.2607 ________________________________________ From: Jingxun Chen Sent: Wednesday, April 27, 2011 1:42 PM To: Elizabeth Y Choe Subject: RE: wiki code

The big picture

• 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 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.
• Project: use Friedland's RTC counter to induce apoptosis in yeast after three replicative cycles

High level ideas

• 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
• 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

Some technical thoughts

• To get more specific, I need to:
  • find guidelines to choose G1 cdk, Promoter X, and Molecule A
  • test the constructs piece by piece and as a whole (characterized by transfer functions)
  • assemble the constructs in yeast
  • ensure that adding the plasmids don't severely lower cell viability
  • find a way to measure apoptosis in yeast (should have method publications)
  • label the yeast cells with biotin that indicate the number of division