Lab meeting presented on February 19th, 2010

2. Lab MeetingFebruary 19th, 2010

3. BN1071, normal binding Binding at 4 C

4. BN1071, blocked binding

5. BN1071, “uptake” binding

6. BN1071 final results

7. Conclusion When fepA is expressed from the chromosome, every receptor is actively transporting the siderophore fepD fepG fepB

8. Uptake Binding FepB

9. Uptake Binding FepD

10. Uptake Binding FepG

11. ENERGY POISONS CCCP added to 0.5mM Incubated for 20 min at 37 C

12. FLUORESCEIN LABELING

13. Post-binding wash Grow cells to OD = 0.9 Spin 10E8 cells in microfuge – chill down Add 100nM 59Fe-enterobactin Incubate 2 minutes at 4 C Spin down cells Resuspend in 100 uL cold MOPS Add 900 uL MOPS at 42 C Incubate 1 minute at 37 C Remove 100 uL aliquots to 25 mL cold LiCl 0.9% and filter

14. Post-binding Wash

15. BN1071, OKN1 and OKN4 get 0.5uM 59Fe for 40 minutes, then 56Fe is added To 10uM. Aliquots are filtered, washed, and counted BN1071 = open circles OKN1 = dark circles OKN4 = open triangles

16. BN1071 = open circles large OKN422 = small circles (black and white) OKN4 = dotted line

18. PUB for OKN422