Quorum Sensing

1. Quorum Sensing iGEM2007

2. Target (eg Nickel) luxR luxI luxI luxR OHHL

4. Bba_F2620 Bba_E0240

6. F1610 - 3OC6HSL Sender Device Produces LuxI enzyme, which produces 3OC6HSL • Sent out for sequencing. Sequence ~300bp. Actual part is 798 bp. • Sequence contains terminator and something (?) after it

7. Colony PCR 500 bp

8. Digest gel • Lane 1: ladder • Lane 2: XbaI/PstI digest of F1610 • Again, band is at 300. 300

9. I3263: Lux Receiver, HSL & R0063 driven • Sent out for sequencing, first and last 650 bp correct • Transformed into BL21, plated onto agar+ampicillin plates

10. Testing I3263 and induction with HSL in BL21 cells • 9 samples and 1 control • Control = cells without induction by HSL • Grew cells for ~4 hours (varied depending on OD readings during growth) • Induced with HSL at 4-, 2-, and 1-hr before flow cytometry appointment • Used different concentrations of OHL: 1nM, 10nM, 100nM • Effective results from related papers were found at around ~9nM

11. Flow Cytometry • Essentially inconclusive - hardly any fluorescence • In future… • smaller density of cells, construct growth curve for bacteria with more accurate calculations/Ods • larger samples volumes • need positive control (constitutive YFP) • Use OHHL instead of N-butyryl HSL

12. Constitutive positive controls • I13522: constitutive GFP • Appeared green under microscope • I5311: YFP induced by IPTG • Appeared green under microscope • J04430: GFP induced by IPTG • Did not fluoresce

13. Parts we’re thinking of using luxR-HSL complex turns off luxpL promoter, so we are seeking another constitutive promoter • R0040 - promoter (tetR, negative) • Action inhibited by addition of tetracycline • R0051 - promoter (lambda cI regulated) • cI binding results in repression of transcription • R0011 - promoter (lacI regulated, lambda pL hybrid) • IPTG-inducible

14. T9002 -GFP Producer Controlled by OHHL Receiver Device • Sending for Sequencing • Testing for fluorescence after OHHL induction

15. Planned Work