Quorum Sensing

iGEM2007

Target (eg Nickel)

luxIluxI

OHHL

luxRluxR

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Bba_F2620 Bba_E0240

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

Colony PCR

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500 bp

Digest gel

• Lane 1: ladder • Lane 2: XbaI/PstI

digest of F1610• Again, band is at 300.

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300

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

• 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

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