Extending theophylline application

How can the caffeine disk results be extended? What experiments can be done?

Redo original experiment to verify results

Natural selection experiment: Plate all clones on one plate and then plate clones in groups of 5 on 4 plates to see if there are other natural factors affecting growth that we haven’t considered.

Finding the best clone: Plate clones in groups of 5 on 4 plates in the presence of caffeine (varying levels for varying experiments). Take the best clone from each plate and plate them on one plate to see the best (all taking into account any additional natural variables that need to be monitored or accounted for).

Broth experiment: If one clone really is the best, then test it in broth to make the experiment/results more versatile. If it is the best, it should dominate the broth very quickly.

Optimal caffeine concentration on plates (Jon Lim)

Questions:

What if the best fitness module is in a naturally failing e coli (due to factors we have not considered or measured)?

Should our future experiments focus on optimization (if we already have a good system) or on diversification (broth, other common lab situations that would be easier for someone to use than plates, etc). Do we care which is best or do we want an easy process that is sufficiently good?

Is more prep time worth a better result (financially)?

Is there a way to figure out which will be more dominant even when it isn’t dominant yet? Is there some sort of trend or flag that we can identify that would indicate a population that will eventually be dominant?

Determine growth in the Ecoli strain

-Wild type in broth at 37 degrees Celsius -Grow them 12-14 hours

The superstar combination #14

2 tests in each campus

1st test tube- broth, tet, chlor and amp 2nd test tube- broth, tet, chlor, amp and

-Broth, tet -Growth with theophylline, tet resistance -no growth, demetylase problem

Question is growth

Two test-tube with all 24 clones

	1st tube- tet and amp

2nd tube tet, chlor and amp

6 tubes of all combinations chlor not run on the last chaperone group

Theophylline control

1Liter LB+4mM 1mL(50ug/mL Amp) 4mL(5mg/mL Tet) 1mL(35ug/mL Chloro)

Cells- 120 mL cells each time