1. Caffeine Disk Results Spring 2014

2. Procedure Grow clone from glycerol stock – Amp or amp + chlor – 4 mM caffeine Measure A590 and standardize to 0.1 or 0.01 Spread 50 ul on Tet only plate with beads Prepare disks – Commercial disks – 35 ul solution per disk – Up to 100 mM caffeine Place disks – Use sterile insulin needle and sterile tip Incubate – 37 C overnight, then room temp for several days

3. Amp Plasmid J119346 – High promoter – High C-dog – RFP

4. Amp Plasmid J119347 – Low promoter – Low C-dog – GFP

5. 20 Clones Amp Plasmid – Promoter – C dog High-High versus Low-Low – Origin pSB1A2 High copy number J119310 Low copy number Chlor plasmid, pSB4C5 – Chaperones pG-Tf2 pTf16 pG-KJE8 pGro7 pKJE7

6. Too many bacteria, A590 = 1.0

7. RFP on All 20

8. Origin PCR All are pSB1A2 All are J119310 with 500 bp with 750 bp

9. Chaperone PCR Marker 1.pG-Tf2 2.pTf16 3.pG-KJE8 4.pGro7 5.pKJE7

10. Procedure Do Caffeine disk experiments with 0, 20, 40, 60, 100 mM Take Pictures Use loop to take colonies from near the disks and streak onto plates for colony isolation Pick individual colonies and measure – RFP on Cytation – Chaperone PCR – Origin PCR

11. All 20 Clones

12. High-High vs. Low-Low

13. Origin PCR

14. Chaperone PCR

15. Genotypes CloneHigh-HighLow-Low1A2J310pG-Tf2pTf16pG-KJE8pGro7PKJE7 1Xxx 2Xxx 3Xxx 4Xxx 5Xxx 6Xxx 7Xxx 8Xxx 9Xxx 10Xxx 11Xxx 12Xxx 13Xxx 14Xxx 15Xxx 16Xxx 17Xxx 18Xxx 19xxx 20Xxx 21Xxx 22Xxx 23Xxx 24xxx 25xxx

16. Sorted Genotypes CloneHigh-HighLow-Low1A2J310pG-Tf2pTf16pG-KJE8pGro7PKJE7 9xxx#14 11xxx 14xxx 15xxx 16xxx 17xxx 18xxx 20xxx 22xxx 23xxx 24xxx 1xxx 2xxx 3xxx 5xxx 7xxx 8xxx 10xxx 12xxx 13xxx 21xxx 6xxx#12 4xxxx#12 + #14 19xxx#7 25xxx#4

17. Grown only overnight at 37 C

18. 1 + 20 with Caffeine Disks

21. Fitness Clocks

22. 1 + 20 with Caffeine Disks 0 mM 20 nM 40 mM 60 mM 80 mM 100 mM

23. Biosensor Expts

24. Too many bacteria, A590 = 1.0

25. RFP and GFP on All 20

26. 1 + 20 with Theo Disks