Additional Ideas for Future Programmed Evolution Research

1. New System-

New Riboswitch, Multistep

-Ex: Adenosylcobalamin riboswitch- This is possibly a good multi-step pathway, but E. coli metabolism may be too centered on the cobalamin system, making it a concern to disrupt the system and not a good option for further research. A system similar to this multi-step pathway would be a great option, as long as the pathway would not disrupt metabolism and have an ill-effect on the survival and regrowth of the bacteria. During discussion, both ideas for a new system were related by being in the vitamin family, due to the articles found while researching. So this may be an indicator to take a more heterologous approach while searching for a new system and riboswitch that would be more foreign and non-dependent for the E. coli metabolism.

-Ex:Thiamine pyrophosphate riboswitch File:Thiamine Pyrophosphate Riboswitch.pdf

In E.coli, thiamine monophosphate is converted to thiamine which then could be converted to thiamine pyrophosphate by thiamine kinase. For this particular riboswitch when thiamine pyrophosphate (TPP) binds to the riboswitch, it will stop transcription. A discussion about the thiamine pyrophosphate riboswitch resulted in the decision to not pursue this specific riboswitch because E.coli relies on this metabolic system to survive and it could be impossible to remove thiamine and still have them survive. This riboswitch is a great example of a multi-step metabolic pathway, but unfortunately it would be too detrimental to remove thiamine from E.coli’s system. It is still a goal to research a multi-step pathway as fascinating as the thiamine->>thiamine pyrophosphate, but still remain similar and practical to the caffeine->theophylline system.

2. Biosynthesis of Caffeine then Theophylline-

•Making our single-step pathway to a multi-step pathway

3. Manipulating the promoter, RBS, origin, and chaperone-

•Ex: 5 promoters and 5 RBS’s or 500 promoters and 500 RBS’s

4. A. Measure clone distribution around different caffeine concentrations (disks)-

•Coloring the clock

4. B. Measure clone distribution on different caffeine concentrations (plates)-

•Ex: 2mM Caffeine -> clone #23 or 4mM caffeine-> clone ?
•Cranking up selection