IGEM:Stanford/2009/Project Homeostasis/Yanofsky1

Recap of 6/29 Yanofsky Meeting
Dear Drew and Christina:

I just wanted to email you with a status update on our meeting with Prof. Yanofsky about the anti-inflammatory device and the trp operon. Today, we discussed the possibility of both modifying the trp operon itself to have greater sensitivity and using an additional signal like kynurenic acid and its corresponding glutamate NMDA receptor site to sense an increase in Treg populations. Prof. Yanofsky suggested that the use of tryptophan as a signal would be the most ideal since cellular transport of tryptophan is better characterized than any other molecule in kynurenine pathway. However, in the event that we are unable to use tryptophan, Yanofsky suggested looking into an intermediate byproduct of the L-tryptophan degradation reaction, anthranilic acid. Prof. Yanofsky stated that anthranilic acid can be metabolized by E. coli but we still need to work out appropriate receptors in the event that we choose to use anthranilic acid as a marker.

The bulk of the meeting with Prof. Yanofsky was spent brainstorming ways tryptophan could still be used. I have listed the ideas in order of the quality and feasibility.


 * 1) Use of Tryptophan analogues: Prof. Yanofsky mentioned that several analogues of tryptophan including 5-methyl tryptophan and 7-aza tryptophan are capable of binding to the trp operon.  Given this, the team is currently exploring the possibility of using tryptophan analogues as opposed to tryptophan as the substrate for IDO.  Using tryptophan analogues would allow us to artificially control the concentration of IDO substrate without significantly changing the architecture of device.
 * 2) Artificial Trp regulator: During the meeting, Prof. Yanofsky mentioned that it was possible to control the rate of cellular metabolism of tryptophan.  The general idea would be to engineer a second E. coli device to rapidly degrade tryptophan so that a constant tryptophan microenvironment around the device is maintained.  Then, any decrease in tryptophan as a result of IDO could be more easily detected by the cell.
 * 3) TrpR binding affinity and Transport: One suggestion from Prof. Yanofsky that could be incorporated into either project (1) or (2) is changing the binding affinity of the TrpR protein so that it has a lower affinity for tryptophan cofactor.  The trpR protein would then require greater concentrations of tryptophan (presumably more than the background levels of tryptophan) to turn the device on.
 * 4) Use of galectin as a signal: Galectin, a member of the lectin family, is constitutively expressed in human CD25+ Treg cells.  Galectin binds to beta-galactoside.  Thus, having a promoter sequence that detects changes in the level of beta-galactoside is a another marker option.

We will be providing greater details about each of these ideas tomorrow at the meeting.

Thanks again, Anusuya