Part I13208: AiiA Enzyme Test
The motivation behind testing this to part is to test the Zinc Hypothesis, and to test qualitatively if AiiA is hydrolysing AHL.
Materials & Equipment
- Wallac Victor 3 Multi-Well Fluorimeter
- Ependorf Tubes
- Gilson Pippettes
- 37oC Shaker
- GFP Standard Solution
- E.coli Growth Medium w/Ampicilin (LB/M9)
- E.coli DH5a Culture Containing I13208
- Inoculate two cultures each from 10ul of stored I13208 in 2ml of both LB and M9 growth medium containing 50ug/ml Ampicilin.
- Incubate at 37oC for overnight in a shaker.
- Following day, prewarm LB/M9 to 37oC by placing in the 37oC incubator
- Measure and record OD600_1 in report sheet
- Inoculate a 2ml fresh culture from the o/n to bring back the OD600 to 0.1, use prewarmed LB/M9 + Ampicilin.
- Volume used to inoculate new culture = (0.1/OD600_1)*2ml
- Return LB/M9 to incubator
- Incubate new cultures at 37oC for 2 hours in a shaker - This returns cells to exponential phase
- Measure and record the OD600_2 in report sheet
- Dilute again for an OD of 0.1 in a new culture of 10ml of a prewarmed LB/M9 + Ampicilin.
- Volume used to inoculate new culture = (0.1/OD600_2)*10ml
- Vortex new I13208 cultures.
- Pipette 990μL of each of the two cultures into 4 labelled ependorf tubes
- To the +IPTG ependorfs add 1μL IPTG
- To the +ZN ependorfs add 1μL Zinc Chlroide
- Add 10μL of 500nM AHL to all tubes