BioSketchHarvard iGEM 2005 3 Testing the UV Pen Energy output: 2mW = ~70 J/m2/s @ ~1.5cm from agar surface Killing curve: Method Plate 100ul 1:10^4 dilution of cells @ OD600 = 0.5 At this density, the colonies are just short of forming a lawn Irradiate marked locations with UV pen for 0, 1, 2, 4, 8, 16, 32, 64s Results Expected: fewer colonies with increasing exposure time Observed: no difference with exposure time Still try to get UV induction? Get another pen, with higher energy output?
BioSketchHarvard iGEM 2005 5 What has been done: CollinsMod Reproducing results of Kobayashi et al. (2004) w. the Collins switch Testing the ThermoSwitches for heat-repression Cloning experiments
BioSketchHarvard iGEM 2005 6 UV Induces Signal in Collins Switch 0J/m26J/m212J/m224J/m248J/m2 Collins switch JM 2.300 GFP reporter Day 1: 37C Day 2: 2mM IPTG Day 3: UV irradiation, o.n. @ 37C Day 4: Assay
BioSketchHarvard iGEM 2005 7 Testing the ThermoSwitches The Circuit: Treatment: 1. o.n. @ 30C 2. o.n. 2mM IPTG, 30C or 0mM, 37/40C 3. assay after 2h or o.n. @ 30C PL*PL*PL*PL*lacIts cI cI P trc gfpmut3b PL*PL*PL*PL*UVHeat/IPTG
BioSketchHarvard iGEM 2005 8 IPTG Modestly Represses Signal in Switches Collins switch 40C switch 37C switch GFP reporter JM 2.300 Untreated 1 day 2h Note: Cells were treated with IPTG overnight; time (2h or 1 day) refers to amount of time after treatment.
BioSketchHarvard iGEM 2005 9 Heat Represses Signal in TS Switches 40C switch 37C switch GFP reporter JM 2.300 Untreated 1 day 2h Note: Cells were incubated at appropriately high temperatures overnight; time (2h or 1 day) refers to amount of time after high-temperature incubation. after high-temperature incubation.
BioSketchHarvard iGEM 2005 10 Summary of Results Collins Switch Default state: OFF IPTG appears to repress reporter below baseline Positive correlation between UV intensity (up to 24J/m2) and % cells ON We have qualitatively reproduced the features presented in Kobayashi et al (2003)! ThermoSwitches Default states: ON IPTG modestly represses reporter expression Heat significantly represses reporter expression, especially for the 40C switch The OFF state is not maintained over time, however
BioSketchHarvard iGEM 2005 11 Speculations On the Lack of Bistability ThermoSwitch can be turned OFF @ 40/37C CI is capable of repressing GFP, which is on a high-copy plasmid LacIts inactivates under heat treatment ThermoSwitch returns to basal ON @ 30C CI is not sufficiently repressing LacIts LacIts promoters are stronger than that of CI LacIts241 and 265 are very efficient @ 30C than CI Collins Switch is basally OFF @ 37C Can LacIts be more efficient @ 30C than LacI is @ 37C? Are there temperatures @ which the ThermoSwitches exhibit bistability? Are there mutations in the promoters of LacIts that affect CI repression? Mutations not present in Collins Switch plasmids nor promoter of GFP. Had to have arisen independently in both ThermoSwitches.
BioSketchHarvard iGEM 2005 12 Finding a Temperature for Bistability Strain Day 1: Cold Day 2: Cold/Heat Day 3: Basal Day 4: Assay 40C switch 30C30C30C 32C 35C 37C 40C 40C30C 32C 35C 37C 40C 37C switch 30C30C30C 32C 35C 37C 37C30C 32C 35C 37C Note: Treatments are for overnight.