Quorum Sensing iGEM2007

Target (eg Nickel) luxI OHHL luxR

Bba_F2620 Bba_E0240

F OC6HSL 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

Colony PCR 500 bp

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

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

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

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

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

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

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

Planned Work